1st Jan 1970

Example loaded in

Participants:

• Tim

Protocols:

• Testing protocol

Experimental:

Procedure:

1. Put example in
2. See if something works
3. Forget to remove it

2nd Jan 1970

Example getting removed

Participants:

• Tim

Protocols:

• Undoing my mistakes protocol

Experimental:

Procedure:

1. Realise you forgot to remove example
2. Remove it
3. Commit, push, merge

16th of May 2024

Participants:

• Henriete
• Vasilis

Experimental:

(1st part) First PCR:

1. The reverse primer was diluted to 100 uM by adding 235 ul of miliq and made stock solution.
2. 100 ul of the stock solution was diluted to 10 uM by adding 90 ul of Miliq to an epp and added 10 ul of F8.
3. 83.25 ul of MiliQ water was added to 3 epps.
4. 11.25 ul of 10x PCR buffer was added to each of the 3 epps.
5. 11.25 ul of 2 mM dNTPs was added to each of the 3 epps.
6. 2.35 ul of 10 mM forward (T7 terminator) and 2.25 ul of 10 mM reverse primer (F8 primer) was added.
7. 1 ul of F8 DNA template was added into the reaction epp.
8. 1 ul of positive control (PET 28 P450 BM3 No2).
9. 1 ul of Mili Q was added to the negative control epp.
10. 2.25 ul of Pfu polymerase was added to the 3 epps.

24th of May 2024

Participants:

• Henriete
• Vasilis

Experimental:

(2nd part) gel electrophoresis:

1. We dissolved the stock solution of 10x TBE to 0.5x TBE by adding 50mL to a measuring cylinder and then adding 950mL demi water to a 1L measuring cylinder.
2. We dissolved 0.9g of agarose into 100 mL of 0.5x TBE.
3. The agarose solution was dissolved until clear.
4. We added 5μl of ethidium bromide to the agarose solution.
5. We poured the agarose solution to the casting tray.
6. We prepared our samples by adding 10μL of the positive, negative and reaction.
7. 2 ul of loading dye (6X) was added to the samples for a total volume of 12 ul.
8. We added the gel to the apparatus and filled it with the buffer until submerged.
9. We added 5 ul of DNA ruler in the 1st and 5th well, and then 12ul of positive, reaction and negative control in 2nd, 3rd and 4th well, respectively.
10. We ran the gel at 110 V for 50 min.
11. We inspected the gel

3rd of June 2024

Participants:

• Vasilis
• Thijmen

Experimental:

(3rd part) DNA extraction from gel:

1. We prepared the reaction sample to run another gel, by adding 20 ul of the 6x dye to the 100 ul sample.
2. We loaded 5 ul of the DNA ladder to the 6 th well, and 2 wells of 40 ul aliquot of the sample to the 7th and 8th wells respectively. We also filled the remaining 20 ul of the sample to the 9th well.
3. We run the gel at 110 V for 1 hour.
4. We cut 4 bands of the factor 8.
5. We weighted the gel with the DNA with a weight of 0.276 (sample 1) and 0.238 g (sample 2).
6. We added 828 ul of Buffer QG and 714 ul to the 2 Epps respectively.
7. The gel and the buffer was vortexed until dissolved.
8. 1 volume of isopropanol was added to the mixture in both epps.
9. The mixtures we added to spin columns and centrifuged for 1 min.
10. The flow-through was discarded and more mixture was added.
11. This was repeated until all of the mixture was centrifuged.
12. 750 ul of buffer PE was added to the column and was centrifuged.
13. The DNA was eluted with 50 ul of mili q water.

Results and Discussion:

Figure 1. Gel, 24/05/24

We can see little smears and some nice bands, it’s not perfect but based on these results we decided to extract the DNA out of the gel and amplify it in order to get a lot of ‘pure’ factor 8 DNA.

Figure 2. 1st DNA ladder, 2nd negative control, 3rd positive, 4th PCR reaction with new primers (RAD102), 5th eluted DNA, 6st another DNA ladder. 03/06/24

The purified DNA gives a clear band at the expected height, but the concentration is too low to continue with it. In addition, the controls don’t look right: the negative control shows a smear while the positive control does not show anything.

Conclusion: New attempts of DNA amplification are needed, new set of primers should be used and the results should be compared.

3rd of June 2024

Participants:

• Henriete
• Maks
• Vasilis

Experimental:

PCR:

1. The forward primer was dissolved.
2. 83.25 ul of miliQ water was added to all samples
3. The current samples are 1 positive, 2 negative, 4 reactions (2 were made using the first f8 template and 2 using the eluted DNA from the gel). Both primers were used this time.
4. 11.25 ul of 10 x PCR buffer was added to all samples.
5. 11.25 ul of dNTPs was added to all.
6. 2.25 ul of positive control (T7 promoter and T7 terminator) was added.
7. For the negative control (1) T7 promoter and F8 negative primer was added and for negative control (2) f8 reverse and forward.
8. For reaction samples for P1 (first set of primers) T7 promoter and F8 reverse were added.
9. For reaction samples for P2 (second set of primers ) F8 reverse and forward were added.
10. 1 ul of PET 28 to the positive control was added.
11. 1ul of Miliq were added to the negative (1) and negative (2) was added.
12. 1ul of eluted F8 into the P1 and P2 was added.
13. 1ul of pcDNA F8 into P1 and P2 was added.

Gel Electrophoresis:

1. The stock solution of 10x TBE was dissolved to 0.5x TBE by adding 50ml of 10x TBE to 950 ml demi water.
2. 0.7 g of agarose were dissolved in 100ml of 0.5x TBE.
3. The agarose solution was dissolved until clear.
4. 5 ul of ethidium bromide was added to the agarose solution.
5. The solution was poured into the tray.
6. The samples were prepared by adding 10ul of the negative, positive, reaction and the eluted DNA and 2ul of 6x loading dye (total volume 12ul).
7. The gel slots were filled like this: 1st DNA ladder, 2nd negative control, 3rd positive, 4th reaction, 5th eluted DNA, 6th another DNA ladder.
8. Note: the tray was filled after we added the first 2 samples.
9. (Discussion:
10. Question - These samples are the 1st DNA ladder and negative sample? Or the negative sample and positive sample?
11. Answer: I believe 1st DNA ladder and negative sample, not entirely sure. We forgot to fill up the tray before adding the ladder/sample to the gel.)
12. The gel was run for 50 min at 110 V.
13. The gel was inspected.
14. To the positive and negative control we added the new primer.

6th of June 2024

Participants:

• Maks
• Thijmen

Experimental:

Electrophoresis:

• 2uL loading dye and 10uL sample (vortexed) were added to a new clean tube. Each sample was briefly vortexed and added to the gel in the following order: ladder, p2 eluted, p1 eluted, positive control, p1 cdna, p2 negative control, p2 cdna, p1 negative and at last again a ladder.
• The gel was run at 110 volts, 50 min

Results and Discussion:

Figure: 1st DNA ladder, 2nd negative control, 3rd positive, 4th reaction, 5th eluted DNA (RAD101), 6st another DNA ladder. 03/06/24

There was no band in the positive control as not the correct primer was used. The negative control had a smear. The reaction had a smear and a small band. The eluted DNA was not amplified, thus there was a small band as expected.

Figure 2. Left to right: 1st DNA ladder, p2 eluted, p1 eluted, positive control, p1 cdna, p2 negative control, p2 cdna, p1 negative and at last again a ladder. 06/06/2024

Again very big smears, molecular ladder was not good visible, only good well was the positive control. For some unknown reason, these samples have not run far on the gel, so the results are difficult to interpret.

Advice:

Column purifies sample 4 and 5. Another suggestion is to restart and make all samples again. Samples 4 and 5 are the 2 samples with good bands on the right site of the gel (you can also count the slots/bands from left to right and then you will see that those are the 4th and 5th).

Conclusion:

We decided to remove the smeared DNA by the means of purification in the next experiment, RAD103. The insight after the purification will provide understanding of such behavior of the DNA.

10th of June 2024

Participants:

• Henriete
• Thijmen
• Vasilis

Experimental:

Purification of PCR product:

1. For the PCR purification, the protocol QIA PCR purification was followed ( “QIAquick® PCR Purification Kit”)
2. For the QI quick purification samples were noted as + con (positive control), P1 cDNA (Plasmid DNA from the frst primers) and P2 -con (Negative control using second set of primers). P2 - con was used since there was a band.
3. 60 microliters of the negative control was pippeted into the QIAquick column after this 300 microliter of PB buffer was added. For the P1 cDNA and positive control samples 90 microliters was pippeted into the QIAquick collumn and after this 450 microliters of PB buffer was added.
4. Next the samples were centrifuged for 60 seconds on 13.000 RPM.
5. The flow-through was discarded.
6. 750 uL PE buffer was added and the samples were centrifuged for 60 seconds at 13000 RPM.
7. The flow-through was discarded and the samples were centrifuged again for 60 seconds.
8. the QIAquick tubes were put into 1.5 ml epps.
9. Next, there was 50 microliters of miliQ water added to the QIAquick membrane for each sample.
10. Samples are centrifuged again for 1 minute.

Used protocol:

PCR of purified PCR product:

3 samples were prepared: positive control (not the purified PCR ‘+’ control) and 2 samples of plasmid cDNA from the purified PCR product (see, 10/06/2024 purification of PCR product).

1. PCR mix was prepared (per sample):
   • 83.25 μL MilliQ water
   • 11.25 μL 10x PCR buffer
   • 11.25 μL 2 mM dNTPs (10 mM each dATP, dTTP, dGTP. dCTP)
   • 2.25 μL 10 μM forward primer (T7 promoter sequence)
   • 2.25 μL 10 μM reverse primer react (1st reverse primer prepared)
2. 1 uL of the PCR purified sample DNA were added: PET 28 as positive control and plasmid cDNA for reaction vials.
3. 2.25 Pfu polymerase was added per sample.
4. the thermocycler was set 51 degrees for 20 cycles.
5. the samples were collected the next day (11.06) at 15:10.

Making of the gel & electrophoresis:

Agarose gel:

1. The stock solution 10x TBE was dissolved to 0.5x TBE by adding 50ml of 10x TBE to 950 ml demi water.
2. 0.7 g of agarose were dissolved in 100ml of 0.5 xTBE.
3. The agarose solution was dissolved until clear.
4. 5 ul of ethidium bromide was added to the agarose solution.
5. The solution was poured into the tray.
6. The samples were prepared by adding 1ul of loading dye to 5 ul of the 3 samples which are positive control, p2 negative control and P1 cDNA. P2 negative control contained DNA band at approximately 7.5 kB and that’s why it was used for purification.
7. For negative control, 5 ul of milliQ was added with 1 ul loading dye.
8. Samples were pipetted in the following way: 1st slot: 5ul DNA ladder, 2nd: 5 ul of p2 negative control (containing DNA), 3rd: 6ul of positive control, 4th: 6 ul p1 cDNA, 5th: 6ul of negative control (5ul of MiliQ +1ul loading dye).
9. Note: The first 2 slots were punctured.
10. The gel was run at 110V for 50 min.
11. The gel was inspected.

28th of June 2024

Participants:

• Thijmen
• Vasilis

Experimental:

Gel electrophoresis of 2 cdna:

1. The stock solution of 10x TBE was dissolved to 0.5x TBE by adding 50ml of 10x TBE to 950 ml demi water.
2. 0.7 g of agarose were dissolved in 100ml of 0.5 xTBE .
3. The agarose solution was dissolved until clear.
4. 5 ul of ethidium bromide was added to the agarose solution.
5. The solution was poured into the tray.
6. The gene ruler 1kb was used and it was prepared by mixing 100 ul of gene ruler 1kb (25-10000) with 100 ul 6x tritrack loading dye and 400 ul of miliq.
7. The samples were prepared by adding 1ul of loading dye to 5 ul of the 3 samples which are positive control, p1 cDNA and p2 CDNA( it should have no elongation) and a negative control.
8. The gel was filled the following way: 1st was punctured with ladder 5ul DNA ladder, 2nd 5ul of DNA ladder, 3rd 6ul positive control, 4th 6ul p1 cDNA, 5th 6ul p2 CDNA, 6th 6ul negative control and 7th 5ul DNA ladder.
9. The gel was run at 110V for 50 min.
10. The gel was inspected.

NOTE: This note describes how the gel is made on this day. On the same day when the gel has been made the PCR products of RAD103 (5th part) have been loaded onto the gel. Later the PCR product of RAD104 (1st part) has been loaded on the same gel. Results of that can be seen in RAD104 (3rd part).

Results and Discussion:

Figure 3. 28/06/24

In the picture above we can see that our results still have smears which is not what we want, the next time we will perform PCR and look at the product on a gel we will use new primers in the hope we can fix the issue with the smears. Nothing has happened which could potentially influence the results.

Indication of 4 slots (from left to right): gene ladder, negative control P2, positive control, cDNA with primers P1. The 4th slot contains a very slight band indicating the amount of amplified DNA was very scarce. if the picture is zoomed in, a slight band at around 7.5 kB can be seen.

Conclusion:

DNA amplification was unsuccessful as no clear band is visible in the expected range (ca 8000 bp). The smears likely indicate bonding between the primers and the formation of large structures of various sizes. Another primer combination should be tried.

28th of June 2024

Participants:

• Lea
• Thijmen
• Vasilis

Experimental:

GFP; new pcr with new primers:

• We first hydrated the new Factor 8 primers (which are coded as FVIII Fwd new2, FVIII Rvd-PA and FVIII Rvd no PA) and the GFP primers to 100 uM.
• Then made 10uM dilutions with the same label as on the primer vials.
• We then made different Factor 8 primer combinations with old and new primers. For the GFP we used the standard used primers.
• We first made a PCR master mix per primer combination (see table below) as follows:
• GFP DNA dilution: 20ng/uL
• 25 microliters of mastermix 1 (PCR M1) are added to PCR tube 1, after this 1 microliter of F8 DNA (or for PCR tube 6, 10 microliters of 20 ng/uL GFP DNA) was added to the PCR tube after which 2,25 microliters of Pfu polymerase is added to the PCR tube. Now the PCR tube can be placed into the PCR machine. Repeat for all remaining master mixes and PCR tubes and then select the program named f8 comb + GFP and start the PCR.

1st of July 2024

Participants:

• Lea
• Thijmen
• Vasilis

Experimental:

Gel electrophoresis:

1. The samples were prepared, by adding 1ul of loading dye to 5ul of the samples.
   • 1st sample: FVIII Fwd new2 + FVIII Rvd,
   • 2nd: FVIII Fwd new2 + FVIII Rvd-PA,
   • 3rd: FVIII Fwd new2 + FVIII Rvd no PA,
   • 4th : FVIII Fwd + FVIII Rvd-PA,
   • 5th:FVIII Fwd + FVIII Rvd no PA,
   • 6th:GFP Fwd + GFP Rvd
   (see 28/06 new PCR with new primers for more info).
2. The gel was filled the following way:
   • 8th slot: 5ul of DNA ladder
   • 9th: 6ul 1st sample
   • 10th: 6ul 2nd sample
   • 11th: 6ul 3rd sample
   • 12th: 6 ul 4rd sample
   • 13th: 6ul 5th sample
   • 14th: 6ul 6th sample
   • 15th: 5ul of DNA ladder.
   Something went wrong with the 4th one - it disappeared. (Discussion needs to be done.)
3. After this, the gel electrophoresis was performed where it was run for 50 minutes on 110 V.

Results and Discussion:

Figure 1. Gel electrophoresis

As seen in the picture above we again got smears on our gel. Another strange anomaly is that in the GFP well (second to last) we could see a very vague band all the way at the bottom of the gel and a very vague band somewhere at the top. This of course shouldn’t be happening since the gel should show 1 band somewhere low in the gel.

For the 5 Factor 8 samples the same could have gone wrong as with all the other PCR reactions that have been performed (we still don’t know what it is), however it is very strange that also the GFP (second to last slot) has also failed to give a good result. We have used the primers that normally work well so it can’t be the primers fault, this might indicate that something is wrong with the procedure which causes the smears.

Conclusion:

We will have to meetup with Frank in order to discuss these results. The reliability of our results is probably good enough, however for the 4th sample the pipetting went a bit wrong which led to the loss of some of the sample (it disappeared in the gel) furthermore during the transfer of the gel from the gel electrophoresis machine onto the UV machine, the gel broke. We reassembled it and we could still see things like the molecular ladder therefore we think it hasn’t impacted our results by a lot.

After consultation with Frank, it was decided to try the multiplying F8 DNA with a different method (RAD107) and to try again with the GFP because it had worked before.

3rd of July 2024

Participants:

• Kate
• Lea
• Thijmen

Experimental:

PCR:

1. GFP template was diluted 10x times to concentration of 2ng/ul.
2. In 1 epp.:
3. 25 ul PCR mix. (prepared by the procedure)
4. 1 ul GFP template. (=2 ng)
5. 1 ul DNA poly. (More that required by the procedure, but assumed to be not significant.)
6. The PCR mixture was added to the PCR machine for 10 cycles. 1 cycle: 95C, 95C, 62C, 72C, 72C, 10C.

Gel electrophoresis:

1. 0.7 g of agarose were dissolved in 100ml of 0.5 xTBE.
2. The mixture with the agarose was heated in the microvawe for 5 min.
3. 5 ul ethidium bromide was added.
4. The gel was poured into the tray and solidified in 20 min.
5. 0.5x TBE buffer was prepared.
6. The buffer was added onto the gel, and afterwards the comb was removed.
7. The analysis samples were prepared, by adding 1ul of loading dye to 5ul of the GFP samples. (3 in total)
8. The samples were loaded in the gel:
9. 1st well: DNA ladder (Kate)
10. 2nd well: GFP sample 1 (Lea)
11. 3rd well: GFP sample 2 (Lea)
12. 4th well: GFP sample 3 (Kate)
13. 5th well: DNA ladder (Thijmen)

Results and Discussion:

Figure 1. 1st well: DNA ladder (Kate); 2nd well: GFP sample 1 (Lea); 3rd well: GFP sample 2 (Lea); 4th well: GFP sample 3 (Kate); 5th well: DNA ladder (Thijmen).

A GFP mRNA was correctly received at the 1000 bp region (see the ladder). However, the spot is very dim, so this means that not many DNA was produced.

Conclusion:

The decision was made to redo the PCR to make more DNA. Then, 3 to 4 reactions of 100 ug with 20 ng of the template per 100 ul will need to be prepared, and they will be run for 25 PCR cycles.

After consultation with Frank, it was decided to try the multiplying F8 DNA with a different method (RAD107) and to try again with the GFP because it had worked before.

3rd of July 2024

Participants:

• Lea
• Maks
• Thijmen

Experimental:

PCR:

1. Mastermix was prepared (see calculations).
2. In 1 PCR epp.:
3. 100 ul PCR Mastermix.
4. 1 ul GFP template. (=20 ng) - 1st: 2ng, 2nd: 2ng (pipette tip was not changed), 3rd: 1 ng, 4th: 1 ng (potentially, less mastermix: bubbles/ not correct ammounts/ was not well visible?).
5. 2 ul DNA poly. - was added in the end. - 1st: 2ng, 2nd: 2ng, 3rd: 2 ng (spill), 4th: 2 ng.
6. The PCR mixture was added to the PCR machine for 35 cycles. 1 cycle: 95C, 62C, 72C.
7. The samples were left in the PCR machine at 4C overnight.

Gel electrophoresis:

1. 0.7 g of agarose were dissolved in 100ml of 0.5 xTBE.
2. The mixture with the agarose was heated in the microvawe for 5 min.
3. 5 ul ethidium bromide was added.
4. The gel was poured into the tray and solidified in 20 min.
5. 0.5x TBE buffer was prepared.
6. The buffer was added onto the gel, and afterwards the comb was removed.
7. The analysis samples were prepared, by adding 1ul of loading dye to 5ul of the GFP samples. (3 in total)
8. The samples were loaded in the gel:
9. 1st well: DNA ladder (Kate)
10. 2nd well: GFP sample 1 (Lea)
11. 3rd well: GFP sample 2 (Lea)
12. 4th well: GFP sample 3 (Kate)
13. 5th well: DNA ladder (Thijmen)

Results and Discussion:

Figure 1. 1st well: DNA ladder (Kate); 2nd well: GFP sample 1 (Lea); 3rd well: GFP sample 2 (Lea); 4th well: GFP sample 3 (Kate); 5th well: DNA ladder (Thijmen).

A GFP mRNA was correctly received at the 1000 bp region (see the ladder). However, the spot is very dim, so this means that not many DNA was produced.

Conclusion:

The decision was made to redo the PCR to make more DNA. Then, 3 to 4 reactions of 100 ug with 20 ng of the template per 100 ul will need to be prepared, and they will be run for 25 PCR cycles.

After consultation with Frank, it was decided to try the multiplying F8 DNA with a different method (RAD107) and to try again with the GFP because it had worked before.

4th of July 2024

Participants:

• Lea
• Maks
• Thijmen

Experimental:

Gel electrophoresis:

1. samples were prepaired with 1uL loading dye and 5uL PCR product.
2. 5uL of the samples was loaded on the gel in the following order: DNA ladder, SPILL, normal, normal, X and another DNA ladder.
3. The gel was run at 110V for 50 min.
4. Since no compound was seen, the PCR samples were mixed with ethidium bromide. Observation: the flask shined under the UV light.

Gel electrophoresis 2:

1. Same gel as above was used and the samples are remade (so again 1 ul of loading dye, 5 ul of PCR product)
2. The wells were loaded as follows: DNA ladder, 2, 2, SPIL, 1.
3. The gel was run at 110V for 50 min.

Results and Discussion:

Figure 1. 1st gel of our product: DNA ladder, SPILL, normal, normal, X and another DNA ladder.

Figure 2. 2nd gel of our product: DNA ladder, 2, 2, SPIL, 1.

In the first gell, no DNA was seen, but we wanted to check the presence of DNA, and added the ethidium bromide to one PCR sample. Since the flask shined, this indicated that there is some DNA. Thus, the new PCR was prepared, as well as the samples were loaded onto a new gel again for second analysis. Still, in the second gel no results were seen which indicated that the DNA was indeed not present. Thus, the new experiment was decided to be conducted - RAD108.

Conclusion:

No DNA apparently was present in the sample. It is likely that an error was made and one of the ingredients was not added correctly. This meant that the procedure would need to be repeated.

3rd of July 2024 - 4th of July 2024

Participants:

• Lea
• Maks
• Thijmen

Experimental:

PCR:

1. Mastermix was prepared (see calculations).
2. In 1 PCR epp.:
3. 100 ul PCR Master Mix.
4. 1 ul GFP template. (=20 ng) - 1st: 2ng, 2nd: 2ng (pipette tip was not changed), 3rd: 1 ng, 4th: 1 ng (potentially, less mastermix: bubbles/ not correct amounts/ was not well visible?).
5. 2 ul DNA poly. - was added in the end. - 1st: 2ng, 2nd: 2ng, 3rd: 2 ng (spill), 4th: 2 ng.
6. The samples were left for 25 cycles PCR. 1 cycle: 95C, 62C, 72C.

Gel electrophoresis:

1. 0.7 g of agarose was dissolved in 100ml of 0.5 xTBE.
2. The mixture with the agarose was heated in the microwave for 5 min.
3. 5 ul ethidium bromide was added.
4. The gel was poured into the tray and solidified in 20 min.
5. The 0.5x TBE buffer was added onto the gel, and afterwards the comb was removed.
6. The analysis samples were prepared, by adding 1ul of loading dye to 5ul of the GFP samples. (3 in total)

The samples were loaded in the gel run on 110V for 50 min.

Results and Discussion:

Figure 1. Gel electrophoresis (0.7% w/v agarose) of the PCR samples

All samples showed a clear band at 1000kb, where it is expected.

Conclusion:

It was decided to continue with the purification.

4th of July 2024

Participants:

• Lea
• Maks
• Thijmen

Experimental:

Purification of PCR product:

1. 500 ul buffer for 100 ul PCR sample.
2. The color of the solution did not change.
3. Some acid and base were added to the buffer. However, no change in color was observed.
4. 600 ul of each sample were added to a separate QIAquick column.
5. Next the samples were centrifuged for 60 seconds at 13.000 RPM.
6. The flow-through was discarded.
7. 750 uL PE buffer was added and the samples were centrifuged for 60 seconds at 13000 RPM.
8. The flow-through was discarded.
9. The samples were centrifuged again for 60 seconds.
10. The QIAquick tubes were put into 1.5 ml epps.
11. Next, there were 50 microliters of milliQ water added to the QIAquick membrane for each sample.
12. Samples are centrifuged again for 60 seconds.
13. The sample was labeled GFP cDNA.

Gel electrophoresis of purified product:

1. 3 ul of dye were added to the 15 ul of the sample.
2. The gel was loaded: 5 ul of the loading die; sample.
3. Gel was run for 50 min at 110 V.

Results and Discussion:

Figure 1. Agarose gel of purified product: ladder; sample.

The result of the Gel is good - a little smear at the top probably occurred due to too much DNA concentration. The band is around 1000 bp, which is where you would expect EGFP.

Figure 2. Analysis with nanodrop:

Measured samples of the GFP cDNA showed a concentration of 60.2 ng/ul.

Conclusion:

The pure GFP cDNA sample of 185 ul with concentration 60.2 ng/ul was received. The application of it is to produce mRNA for the further experiments and testing of the vesicles.

3rd of July 2024 - 4th of July 2024

Participants:

• Lea

Experimental:

Procedure:

1. Top10Transf colony was prepared the following way: 1ul FVIII plasmid solution was mixed with 200ul Top10 bacteria suspension.
2. XL1BlueTransf colony was prepared the following way: 1ul FVIII plasmid solution was mixed with 200ul XL1Blue bacteria suspension.
3. Both of the cultures were left on ice for 0.5 h.
4. Both bacteria were heat shocked for 54 seconds at 42C.
5. 800 ul of LB medium.
6. Left in shaker for 0.5h 37C.
7. Bacteria were spun down with a centrifuge and 900uL medium was removed
8. The pallet was resuspended in the remaining 100uL medium and plated on agar plates containing ampicillin.
9. The bacteria were left in the incubator (37 C)overnight.
10. (the next day)
11. after 24 h incubation, the Top10 plate had many miniscule colonies, while Xl1Blue had less, but larger colonies.
12. 6 colonies were picked from the XL1Blue plate and 4 from the Top10 plate.
13. in total 10 5mL liquid cultures were prepared and incubated overnight at 37C.

Conclusion: These successful bacteria synthesis allows for further FVIII isolation and production (RAD110)

5th of July 2024

Participants:

• Kate

Experimental:

Cultures used:

• E. Coli XL1 Blue: 1, 2, 3, 4, 5, 6 cultures with pc DNA Factor VIII
• E. Coli: Top 10 1, 2, 3, 4 cultures with pc DNA Factor VIII

Procedure:

1. 2ml of each bacterial culture was added. Labeled respectively: XL1, XL2, XL3, XL4, XL5, XL6; Top1, Top2, Top3, Top4
2. I centrifuged the pipetted cultures for 3 minutes at 1000 rpm, RT
3. Supernatant was collected leaving pellets dry
4. 2 ml more of of cultures were added to each sample
5. Samples were centrifuged again, same way
6. Supernatant was collected leaving pellets dry
7. 250 ul of P1 buffer was added to all samples
8. To fractions XL1-5 250 ul of P2 buffer were added
9. Samples XL1-5 were inverted 6 times (kept in the buffer P2 less than 5 min)
10. 350 ul of N3 buffer was added to solutions XL1-5, samples were rotated 4-6 times [NOTE: XL2 had too much liquid (~1.25 ml), but pH paper showed that it is okay, so probably N3 buffer was added 2 times)]
11. Same as to XL1-5 was doe to XL6 and Top1-4
12. The samples were put to centrifuge 10 min at 13000 rpm, 20C
13. 800 ul supernatants was collected to QIAprep spin column
14. This supernatant in QIA prep was centrifuged for 30-60 seconds at 13000 rpm, and the flow-though was discarded
15. 250 ul PE buffer were added
16. Centrifuged 60 sec
17. flow-through was discarded
18. 250 ul PE buffer were added
19. Centrifuged 60 sec
20. flow-through was discarded
21. 50 ul H2O MiliQ added
22. Centrifuged 60 sec
23. flow-through was discarded
24. 50 ul H2O MiliQ added

Digestion:

• Combine EcoRI with plasmid for 10 digestions
• Combine XhoI with plasmid for 10 digestions
• From pc DNA FVIII (12086 bp)
• XL1 Blue: 2.5 ul to EcoRI and 2.5 ul to XhoI & Top10 2.5 ul to EcoRI and 2.5 ul to XhoI

Linearisation:

• EcoRI: 4 expected bands to be seen on gel: cuts at 347bp, 1728bp, 4688bp, 5323bp
• XhoI: 1 expected bands to be seen on gel: cuts at 12.1 kbp
• Composition for linearization mixture per one linearization: 2.5 ul buffer, 0.5 ul enzyme, 5 ul DNA, 17 ul MQ
• Sample with enzymes is linearized at 37 C for 2 hours

Analysis:

• Prepare gel
• After linearization, mix with 6ul 6xLB
• Put on gel

Results and Discussion:

Figure 1. EcoRI digest

The EcoRI digestion showed 4 bands, as expected.

Figure 2. XhoI digest.

The Xhol digestion shows one band, as expected.

Figure 3. Expected results scheme

Conclusion: FVIII pcDNA was extracted from the bacterial cultures Top10 and XLBlue. The gel analysis of enzymatically linearized DNA confirmed its proper structure. The further steps of the FVIII DNA processing is the linearization of more plasmid, and then purifying it. This DNA then should be transcribed to FVIII mRNA.

8th of July 2024

Participants:

• Lea

Experimental:

Cultures used:

• E. Coli XL1 Blue: 1, 2, 3, 4, 5, 6 cultures with pc DNA Factor VIII
• E. Coli: Top 10 1, 2, 3, 4 cultures with pc DNA Factor VIII

Procedure:

1. the concentration of all isolated plasmid samples was determined
2. 4 enzyme digestions with a volume of 150 uL were prepared using plasmids isolated from XL1Blue following the protocol. The DNA concentration of the plasmids used was 758 and 756 ng/uL,so 11.9 uL was used to reach 9ng pcDNA.
3. digestion was incubated at 37 C for 3 hours
4. The digested plasmids were purified using a spin column (following the same protocol as before):
5. 600uL PB buffer to 120uL digestion mix, centrifuge for 1 min at 13000 rpm
6. the concentrations of the purified product were measured to be 79.9, 96.6, 94.7 and 88.8 ng/uL in that order
7. the purified products were put on gel (110V 50 min) to check the quality
8. the samples were floating out of the wells, so we prepared them again with a 5:2 sample to dye (double the amount of dye)
9. samples were loaded: ladder-unpurified———middle wells empty———1-2-ladder-3-4

Results and Discussion:

The digested plasmids show clear bands at the length where we expected them.

Conclusion: A large amount of linear FVIII pcDNA was produced and purified. This can now be used to perform in vitro transcription and produce FVIII mRNA.

9th of July 2024

Participants:

• Lea

Experimental:

1. a mastermix for 10 reactions of 20uL was prepared following the protocol for IVT. → ! Guanosine-5’-monophosphate was not used in the reaction mix, because it prohibits capping. mastermix amounts:
   • Tris (1M): 8uL
   • MgCl2 (1M): 5uL
   • DTT(0.1M): 10uL
   • spermidine (25mM): 8uL
   • rNTPs(20mM): 40uL
   • MilliQ: 129uL
   • total: 200uL
2. prepare 10 epps, with each 20uL mastermix
3. 1uL dsDNA of the five different DNA samples (4 FVIII DNA, 1 GFP DNA) was added to epps (for FVIII linearised plasmid, 75 ng of DNA is needed for 20uL reaction, for GFP it is 26ng. The concentration of the FVIII DNA was close enough that 1uL could be taken from the tube. The GFP DNA was diluted 1:1 to a concentration of 30ng/uL and 1uL was used from that )
4. For every different DNA sample, one epp got 2uL T7 polymerase and the other 4uL. → 2uL is the amount recommended by the protocol, but because of the length of the FVIII gene, we wanted to see if a larger amount of T7 would be beneficial.
5. The reaction was incubated at 37 C for 3 hours.
6. 5uL of all samples was mixed with 2uL loading dye and run on an agarose gel (110V 40 min)

Results and Discussion:

Figure 1. Agarose gel electrophoresis of IVT samples. To prevent waste, we use the same agarose gel for multiple experiments. The crossed out lanes are from a previous experiment.

It is weird that 1, 2 and 4 with 2uL T7 did not show a lot of transcription while 3 seems to have a lot, because they should practically be the same. Overall, the reaction seems to work better with 4uL T7. The FVIII RNA was expected to be longer than 7000 instead of 1200. This could be the result of RNA degradation or self splicing, but it could also be because of the properties of the gel and because RNA is not linear. In addition, the GFP RNA did not work out that well in both cases. it could have something to do with the template. maybe the calculations on the amount of template needed was incorrect.

→ I (Lea) recalculated the amount of template needed and I did receive a different outcome: 45 ng/20uL. This is a 15ng difference from what was used in this experiment.

Conclusion: Experiment needs to be repeated with proposed changes:

• different version of T7 poly.
• Add RNAse inhibitor.
• Reduce temperature to prevent autocatalytic breakdown.

10th of July 2024

Participants:

• Lea
• Thijmen

Experimental:

IVT:

1. a 200 uL mastermix was prepared the same as the one in RAD112
2. The mix was divided over 10 epps labeled the same as the descriptions in the table.
3. samples were prepared following the table:
   • 4uL of T7 was used for both versions
   • 0.5 uL of RiboLock RNase inhibitor was used
   • 1 uL of F8 DNA template was used from both template samples, 1.5 uL of the diluted GFP template was used to reach the right amount of template.
4. The reactions were incubated for 3 hours.

Gel electrophoresis:

1. Samples with dye were prepared - 5 ul sample + 2 ul dye.
2. The samples were loaded as follows:

• 1st well: Sample 1,
• 2nt: Sample 2,
• 3th: Sample 3 (see discussion),
• 4th: Sample 4,
• 5th: Sample 5,
• 6th: DNA ladder,
• 7th: Sample 6,
• 8th: Sample 7,
• 9th: Sample 8,
• 10th: Sample 9,
• 11th: Sample 10.

Results and Discussion:

Figure 1. Agarose gel electrophoresis of IVT samples. To prevent waste, we use the same agarose gel for multiple experiments. The crossed out lanes are from a previous experiment.

During the pipetting of the sample 3 the pipette tip fell off, which may be the cause of the absent band. Samples 4 and 5 seem pure with only minor smearing. Samples 2 and 7 have a significant smear, which could be perhaps resolved by using denaturing conditions that would eliminate the RNA’s autocatalytic activity.

Conclusion: Adding an RNase inhibitor improves the outcome of IVT. Sample analysis on denaturing gel should be carried out in order to eliminate autocatalytic activity of the RNA molecule. GFP samples give more clear bands on the native gel than the factor VIII samples.

15th of July 2024

Participants:

• Adrian
• Thijmen

Experimental:

1. a mastermix for 5 reactions of 40uL was prepared following the protocol for IVT. → ! Guanosine-5’-monophosphate was not used in the reaction mix, because it prohibits capping. mastermix amounts:
   • Tris (1M): 8uL
   • MgCl2 (1M): 5uL
   • DTT(0.1M): 10uL
   • spermidine (25mM): 8uL
   • rNTPs(20mM): 40uL
   • MilliQ: 129uL
   • total: 200uL
2. prepare 5 epps, with each 40uL mastermix, labeled with numbers 1 - 5. Note - epp number 5 had an air droplet in the pipette tip, so it's doubtful whether it has the correct amount
3. Factor 8 DNA - 2, Factor 8 DNA 3, GFP diluted were measured on nanodrop for concentration:
4. 
5. The following were added to epps 1 - 5 in order from left to right:
   • 1 = 1.78 ul Factor 8 - 2 + 8 ul polymerase
   • 2 = 1.78 ul Factor 8 - 3 + 8 ul polymerase
   • 3 = 1.78 ul Factor 8 - 2 + 1 ul RNase inhibitor + 8 ul polymerase
   • 4 = 1.78 ul Factor 8 - 3 + 1 ul RNase inhibitor + 8ul polymerase
   • 5 = 2.14 ul GFP DNA + 1 ul RNase inhibitor + 8 ul polymerase

   Note: From calculating the concentration of DNA specified in the manual (10nM) considering that our DNA is roughly 8000 bp which are all roughly 618g/mol each we calculated that the final concentration that manual uses is 49 ng/ul which is way higher than what we have used in all experiments so far.

   Note: Use the mutated polymerase frank gave us (green label)

   Solutions transferred to PCR tubes labeled 1-5 with roman numbers

   Solutions stored overnight in PCR machine at 4 degrees centigrade

6. All 5 products analyzed on a denaturing agarose gel:
   • Added 0.35g agarose in 5 ml 10xMOPS solution and 36 mL MilliQ
   • Heat in microwave for about 15 seconds (until complete dissolution)
   • Stir continuously while slowly adding 9ml 37% formaldehyde
   • Stir for 5 seconds more and pour in casting tray
   • Wait 50 minutes
   • Load samples on gel
   • Run for 50 minutes
   • Analyze under UV

Results and Discussion:

Figure 1.

The samples with inhibitors show more clearly visible bands on the gel. There are significant smears on the gel. Further purification and modification is required in order to obtain a clearer result. There is only one visible band per sample.

Conclusion: The procedure used in this experiment is effective at synthesizing and analyzing mrna. Rnase inhibitors should always be used for better results.

17th of July 2024

Participants:

• Adrian
• Lea
• Thijmen

Experimental:

1. 200uL of master mix was prepared:
   • Tris (1M): 8uL
   • MgCl2 (1M): 5uL
   • DTT(0.1M): 10uL
   • spermidine (25mM): 8uL
   • rNTPs(20mM): 40uL
   • MilliQ: 129uL
   • total: 200uL

   Note: the GFP DNA template made an unexpected sound of ice cracking when taken out of the fridge.

2. The mastermix was distributed over 5 epps giving them 40 uL each.
3. 1uL RiboLock RNase inhibitor was added to all epps
4. 2.14 uL of diluted GFP DNA template was added to epps z (labled GFP RNA on the side)
5. 1.78 uL (for a final concentration of 49ng/uL) of FVIII - 3 DNA template was added to epps 4* and 5* (labled FVIII RNA on the side)
6. 8uL of mutated T7 RNA polymerase was added to every epp.
7. The reaction was incubated at 37C for 3 hours.
8. The samples were checked on a denaturating agarose gel (40min 110V).

Results and Discussion:

The bands all show up at the same hight, while a significant difference in length was expected. the first 3 samples were made with GFP template DNA and the last 2 with F8, so the first 3 should be way lower compared to the last 2. Although we were quite confident in our pipetting, this could be due to a pipetting mistake while adding the template DNA or when putting the samples on the gel. RiboLock RNase inhibitor should always be addded in this procedure.

Conclusion: Only GFP mRNA was rececived & analysed on the gel. Samples 1*, 2*, 3*, 4*, 5*.

Next steps:

purification with phenol chlorophorm extraction.

optimise for GFP, then move on to FVIII.

18th of July 2024

Participants:

• Adrian
• Lea
• Maks
• Thijmen

Experimental:

1. 20uL of eGFP RNA sample 1* (from RAD115) was combined with 20uL Phenol/Chloroform/Isoamyl alcohol (https://www.carlroth.com/nl/nl/a-to-z/rotiphenol-chloroform-isoamyl-alcohol/p/a156.1)
2. shaken vigorously inside fume hood for 15 seconds.
3. centrifuged at 12000G at 4C for 2 min
4. the top aqueous layer was transferred to a new epp and combined with a new 20uL of phenol/chloroform/isoamyl alcohol
5. shaken vigorously inside fume hood for 15 seconds
6. centrifuged at 12000G at 4C for 2 min
7. aqueous layer was transferred to a new epp
8. 50uL of isopropanol was added and shaken
9. centrifuged at 20000G at 4C for 10min
10. the isopropanol was removed and the pellet was washed with 40 uL ice cold isopropanol
11. centrifuged at 25000G at 4C for 5 min
12. The isopropanol was removed and the palate was dried to air.
13. the pellet was resuspended in 10 uL milliQ. sample labeled: RNA pure GFP 18-07-24
14. the concentration of the purified RNA was measured with nanodrop to be 2562 ng/uL

22nd of July 2024

Participants:

• Lea
• Maks

1. To prevent high concentrations of RNA on the gel, the 10 uL sample was diluted with 850uL MilliQ
2. 5uL of the diluted sample was combined with 15uL of denaturing RNA loading buffer, incubated at 65C for 5min
3. the quality of the RNA was investigated by a denaturing agarose gel (110V, 50 min).

the gel showed nothing. possibly because the sample was too diluted.

Results and Discussion:

• Gel on purified product showed no product; problem not in running buffer.
• Maybe, a mistake was made (someone pipetted the wrong fraction), maybe, the sample did not have the mRNA - only single nucleotides.
• Should try to run the gel for 5 minutes.
• Maybe, the reason is that mRNA doesn’t have a cap, it might denature. RNAse might break it down.
• Maybe, we should first modify mRNA, and then purify. - look it up/ ask Frank. 3’-5’ mRNA

Endo or exonuclease which might affect the mRNA.

• Before that - try to work with chemicals from Frank’s lab.
• Maybe, Afka can help you to troubleshoot this purification. (Maks)

Conclusion:The purification procedure does not seem to yield the desired results. Procedure optimisation is required prior to further attempts.

22nd of July 2024

Participants:

• Lea
• Maks

Experimental:

1. we tried to reconcentrate the diluted RNA sample (see RAD116) by precipitating it with 200uL ice cold isopropanol and centrifuging at 27500 rfc at 4C for 5 min → did not do anything
2. to the remaining 20 uL of sample 1* (see RAD115), 20 uL phenol/chloroform was added and shaken.
3. the sample was centrifuged at 12000 rfc, 4C for 2 min
4. the top (aqueous) layer was transported to a new epp together with 20 uL new phenol chloroform and shaken
5. centrifuged at 12000 rfc, 4C for 2 min
6. top layer was added to 50 uL ethanol, shaken
7. centrifuged at 29000 rfc, 4C for 10 min
8. supernatant was removed and the pellet was washed with 40 uL ice cold ethanol.
9. centrifuged at 29000 rfc, 4C for 5 min
10. the supernatant was removed and the pellet was air dried.
11. a small bit of the pellet was removed, and resuspended in 10uL milliQ for analysis
12. the rest of the pellet is stored in the freezer, before use it should be resuspended in milliQ
13. The fragment of the pellet was analyzed on a denaturating agarose gel (110V, 50min)
14. absolutely no bands were observed on the gel

Discussion:

The problem is most likely not caused by a too dilute sample, but it could be the result of the buffer being multiple days old. It might have picked up RNases that have degraded the sample while in the gel.

23rd of July 2024

Participants:

• Maks
• Thijmen

1. Fresh buffer was prepaired
2. the palete was resuspended in 15uL milliQ and the concentration was measured to be 1881 ng/uL.
3. 5uL of the sample was run on a denaturing agarose gel (110V 50min)
4. No bands were discovered.

Discussion:

The problems are not caused by a diluted sample or by an old buffer. It might be a more fundamental problem with the purification method.

Conclusion: Optimization of the purification procedure is needed before the continuation of the experiments as the current procedure seems to yield suboptimal results.

22nd of July 2024

Participants:

• Lea
• Maks

Experimental:

1. 200 uL mastermix was prepared, mixed with 10 uL FVIII - 2 template DNA. the IVT mix was divided over 2 PCR epps and after incubating for 10 minutes, we realized we forgot to add T7
2. 5 uL of WT T7 was added to each PCR tube.
3. the reaction was run in the PCR machine at a constant 37C for 3 hours, then kept at 4C overnight.

23rd of July 2024

Participants:

• Maks
• Thijmen

1. The IVT reactions were removed from the PCR machine and the RNA concentration was measured.
2. The tube with the red marking had a concentration of 7446ng/uL and the one without marking had a concentration of -6389ng/uL even after cleaning and re-blanking.
3. The two samples were run on a denaturing agarose gel (110V 50 min)

Results and Discussion:

The gel showed no bands, likely indicating either a complete samples’ degradation or a mistake made during the purification. The latter idea is supported by the seemingly negative RNA concentration in the sample

Conclusion: New mRNA samples have to be prepared, care should be taken to avoid any potential mistakes during IVT setup or mRNA purification.

29th of July 2024

Participants:

• Lea

Experimental:

The purified GFP mRNA sample was taken out of the fridge after which it was quickly vortexed. In order to have enough sample to be able to pipet it 5ul of miliQ water was added to the sample. After this 5uL of the purified mRNA GFP sample was added to an epp and 2ul of loading dye was added to the same epp. This epp was then vortexed to mix the 2 ingredients. Next, the sample + loading dye mixture was pipetted into the gel after which the gel was run for 5 minutes at 110 volts.

Result

Figure 1. Attempt 1 gel of purified GFP mRNA. 5 minute gel check.

Discussion

As can be seen on the photo, even after 5 minutes of running the gel there is no band visible. This would indicate that the current purification method is not working properly. A new purification method is used in RAD120.

Conclusion: Purification method should be revised, and the new method repeated and performed.

29th of July 2024

Participants:

• Maks
• Thijmen

Note: all the materials that were used during the purification (like epps and pipet points) were sterile and all the work was done with gloves. This all will ensure a RNase free environment.

1. 20uL of a GFP mRNA sample 1 was combined with 20uL Phenol/Chloroform/Isoamyl alcohol
2. shaken vigorously inside the fume hood for 15 seconds.
3. centrifuged at 12000 G at 4C for 2 min
4. the top aqueous layer was transferred to a new epp and combined with a new 20uL of phenol/chloroform/isoamyl alcohol
5. shaken vigorously inside fume hood for 15 seconds
6. centrifuged at 12000 G at 4C for 2 min
7. aqueous layer was transferred to a new epp
8. 50uL of 96% ethanol was added and shaken
9. centrifuged at 25000G at 4C for 2min (this was a mistake and should have been 10 minutes)
10. The ethanol was removed and 50 ul of 70% ethanol solution was added to the sample.
11. centrifuged at 25000G at 4C for 5 min
12. The ethanol was removed and the palate was dried to air.
13. the pellet was resuspended in 10 uL milliQ. sample labeled: Pure GFP 29/07/2024

29th of July 2024

Participants:

• Maks
• Thijmen

The purified GFP mRNA sample named Pure GFP 29/07/2024 was taken. After this 5uL of the purified GFP sample was added to an epp and 2ul of loading dye was added to the same epp. This epp was then vortexed to mix the 2 ingredients. Next, the sample + loading dye mixture was pipetted into the gel after which the gel was run for 5 minutes at 110 volts.

30th of July 2024

Participants:

• Thijmen

The purified GFP mRNA sample named Pure GFP 29/07/2024 was taken. After this 5uL of the purified GFP sample was added to an epp containing 2ul of loading dye. This epp was then vortexed to mix the 2 ingredients. Next, the gel was loaded. In the first well 5 ul of a molecular ladder was pipetted and in the second well 5 ul of the sample + loading dye was pipetted. After this the gel was run for 50 minutes at 110 volts.

30th of July 2024

Participants:

• Thijmen

A new gel was made by using the following protocol:

1. 100 ml of 0.5 xTBE was put in a flask
2. 0.7 g of agarose was dissolved in the 100ml of 0.5 xTBE flask.
3. The solution was then mixed
4. The mixture with the agarose was heated in the microwave until it became clear
5. 5 ul ethidium bromide was added to the mixture.
6. The gel was poured into the tray and solidified by waiting.

After the new gel was made the sample was prepared. The purified GFP mRNA sample named Pure GFP 29/07/2024 was taken. After this 5uL of the purified GFP sample was added to an epp containing 2ul of loading dye. This epp was then vortexed to mix the 2 ingredients. Next, the gel was loaded. In the first well 5 ul of a molecular ladder was pipetted and in the second well 5 ul of the sample + loading dye was pipetted. After this the gel was run for 50 minutes at 110 volts.

Results and Discussion:

We hope that by replacing the isopropanol with ethanol and by working with sterile equipment that the mRNA will survive the purification step and that we will receive pure GFP mRNA. In order to check if the purification really worked we have to run the samples on a gel.

Figure 1. Attempt 2 gel of purified mRNA. 5 minute gel check. Date: 29-07-2024

As can be seen on the figure 1, there is mRNA present in the sample. In order to check if the RNA is pure and if it is the right RNA a complete gel electrophoresis must be done including a molecular ladder.

Figure 2. Gel electrophoresis of pure GFP RNA sample part 1. Date: 30-07-2024

As you can see on the photo the sample looks pure as far as we can see. However both the sample and the ladder are very vague. This could be due to an error, a too low concentration (which would be bad in the case of the sample) or that the gel is too old. It was hypothesized that the gel is too old since it has been made 1 or 2 weeks prior. Next step is to refresh the gel and rerun the sample.

1st of August 2024

Participants:

• Maks

Experimental:

1. 40 uL of a GFP mRNA sample 1 was combined with 40 uL Phenol/Chloroform/Isoamyl alcohol
2. shaken vigorously inside fume hood for 15 seconds.
3. centrifuged at 12000G at 4C for 2 min
4. the top aqueous layer was transferred to a new epp and combined with a new 40 uL of phenol/chloroform/isoamyl alcohol
5. shaken vigorously inside fumehood for 15 seconds
6. centrifuged at 12000G at 4C for 2 min
7. aqueous layer was transferred to a new epp
8. 100 uL of 96% ethanol was added and shaken
9. centrifuged at 21300G at 4C for 2min (this was a mistake and should have been 10 minutes)
10. The ethanol was removed and 100 ul of 70% ethanol solution was added to the sample.
11. centrifuged at 21300G at 4C for 5 min
12. The ethanol was removed and the palate was dried to air.
13. The pellet was resuspended in 50 uL milliQ. sample labeled: GFP, white box, freezer C1 in frank’s lab
14. Steps 1-13 were repeated with 30 uL FVIII mRNA taken from epps 2, 3, 4 (10 uL each)
15. The samples were analyzed on the regular agarose gel (see below)

Results and Discussion:

Figure 1. Gel electrophoresis of the purified samples

A small band is visible for the GFP, however, with a large smear above and below it. Factor 8 did not appear on the gel, however, as it was taken from an old epp, it may have been degraded prior to purification. The likely explanation for the weak gel signal is the unsterile conditions of the pipette tips and epps as they have not been sterilized after previous use, or the old buffer used to run the gel.

Conclusion: More proper sterilization of the equipment is needed prior to every purification procedure. More mRNA of both GFP and factor 8 should be produced.

1st of August 2024

Participants:

• Maks
• Thijmen

Experimental:

We labeled epps 1 to 4 and we used the same protocol as was described in RAD114. Only difference is that we used the WT polymerase instead of the mutated polymerase and also the amount of DNA added to the samples is different due to a lower DNA concentration.

• 1= F8 (4,5 ul) + mut poly (8 ul)
• 2= GFP (5 ul) + mut poly (8 ul)
• 3= GFP (5 ul) + WT poly (8 ul)
• 4= GFP (5 ul) + WT poly (8 ul)
• F8 pcdna 1 t/m 4 (pcdna = plasmid cloning DNA).

Results and Discussion:

Figure 1. Gel electrophoresis of the samples obtained from the IVT

As can be seen, the gel shows no bands, likely indicating a mistake during the preparation of the IVT mixture.

Conclusion: Since there likely was a mistake in the IVT setup, the experiment should be repeated.

2nd of August 2024

Participants:

• Maks
• Thijmen

Experimental:

2* and 3* sample for purification. Same protocol as for RAD116 In both cases, 35 ul of sample and 35 ul phenol/chloroform/isoamyl alcohol mixture were initially combined, 75 uL ethanol were used and in the last step the sample was dissolved in 50 uL Milli-Q water.

Results and Discussion:

Figure 1. Gel electrophoresis of the purified samples

As can be seen, only 2 small bands corresponding to the eGFP mRNA are visible, both containing a smear; and no bands are observed that would correspond to factor 8. The likely explanation for that would be mRNA degradation as a result of using non-sterile equipment when handling the samples.

Conclusion: The mRNA purification was unsuccessful and should be repeated with sterile equipment and solutions.

13th of August 2024

Participants:

• Adrian
• Kate
• Thijmen

Experimental:

• Procedure for purification followed from RAD120
• NOTE: samples vortexed as well as vigorously shaked
• NOTE: Second and third extractions are performed with 80ul phenol/chloroform/alcohol instead of 20ul
• NOTE: 385 ul ethanol in 165 ul milliq to make 67.2% ethanol solution
• NOTE: sample with red dot on label was touched without glove
• NOTE: When the tray with the samples was being put down after the last centrifuge step, it was knocked over and the step had to be repeated
• NOTE: The samples were airdried 30 min
• NOTE: results show only phenol/chloroform present; Further washing with 70% ethanol or extraction with just chloroform can help concentrate the sample

Results

DNA concentration of the samples in ng/uL

• F81 - 225.5
• F82 - 325.7
• F83 - 32
• F84 - 1144.6
• GFP 4* - 27.6
• GFP 5* - 381.2
• Factor 8 unknown - 42.8

Discussion:

It seems that the machine showed the phenol contamination, as well as no mRNA present in the samples. This leads us to the conclusion that we will not test these mRNAs on the gel, since no mRNA was left.

Conclusion: The experiment needs to be repeated in RAD126.

9th of July 2024

Participants:

• Lea

Experimental:

1. a mastermix for 10 reactions of 20uL was prepared following the protocol for IVT. → ! Guanosine-5’-monophosphate was not used in the reaction mix, because it prohibits capping. mastermix amounts:
   • Tris (1M): 8uL
   • MgCl2 (1M): 5uL
   • DTT(0.1M): 10uL
   • spermidine (25mM): 8uL
   • rNTPs(20mM): 40uL
   • MilliQ: 129uL
   • total: 200uL
2. prepare 10 epps, with each 20uL mastermix
3. 1uL dsDNA of the five different DNA samples (4 FVIII DNA, 1 GFP DNA) was added to epps (for FVIII linearised plasmid, 75 ng of DNA is needed for 20uL reaction, for GFP it is 26ng. The concentration of the FVIII DNA was close enough that 1uL could be taken from the tube. The GFP DNA was diluted 1:1 to a concentration of 30ng/uL and 1uL was used from that )
4. For every different DNA sample, one epp got 2uL T7 polymerase and the other 4uL. → 2uL is the amount recommended by the protocol, but because of the length of the FVIII gene, we wanted to see if a larger amount of T7 would be beneficial.
5. The reaction was incubated at 37 C for 3 hours.
6. 5uL of all samples was mixed with 2uL loading dye and run on an agarose gel (110V 40 min)

Results and Discussion:

Figure 1. Agarose gel electrophoresis of IVT samples. To prevent waste, we use the same agarose gel for multiple experiments. The crossed out lanes are from a previous experiment.

It is weird that 1, 2 and 4 with 2uL T7 did not show a lot of transcription while 3 seems to have a lot, because they should practically be the same. Overall, the reaction seems to work better with 4uL T7. The FVIII RNA was expected to be longer than 7000 instead of 1200. This could be the result of RNA degradation or self splicing, but it could also be because of the properties of the gel and because RNA is not linear. In addition, the GFP RNA did not work out that well in both cases. it could have something to do with the template. maybe the calculations on the amount of template needed was incorrect.

→ I (Lea) recalculated the amount of template needed and I did receive a different outcome: 45 ng/20uL. This is a 15ng difference from what was used in this experiment.

Conclusion: Experiment needs to be repeated with proposed changes:

• different version of T7 poly.
• Add RNAse inhibitor.
• Reduce temperature to prevent autocatalytic breakdown.

13th of August 2024

Participants:

• Kate
• Thijmen

Experimental:

• Procedure RAD120.
• NOTE: For epps GFP 5*, F8 4, F8 II, F8 1, GFP V there was not enough material.
• NOTE: Sample 4* opened during the first centrifuge, we think that nothing spilled.
• NOTE: We added to the regular procedure: 20 ul aqueous/ 20ul organic. + 80 ul MiliQ & + 80 uL Phenol/Chloroform/Isoamyl in all of the steps. So, 100 ml aqueous & 100 ml organic - for better separation.
• NOTE: Phenol/Chloroform/Isoamyl mixture was whitish and colloidal. Associated either with how it looks or it went bad.
• NOTE: F8 mRNA (unknown), F8 II, F8 4, 5* had too little of an organic phase.
• NOTE: After 2 steps of Phenol/Chloroform/Isoamyl extraction, we have also done an extraction step with chloroform.
• Note: roman 2 fell on the centrifuge.
• NOTE: Step with 70% ethanol was repeated 2x times.
• No pellet was seen after the centrifuge.
• Samples GFP 4*, 5*, F8 3, F8 1 and unlabelled sample (red dot) dried & 10 ul of MilliQ was added to each.
• Not fully dried samples were left in the freezer overnight.
• NOTE: Sample GFP 4* and F8 3 were accidentally pipetted out.
• NOTE: No liquid was seen in GFP sample 4*.

Results and Discussion:

Figure 1. Gel of all the samples except for 5* GFP, FVIII 1, FVIII 2.

No samples were seen. Since the procedure was repeated twice, the likely reason for no visible bands on the gel is mRNA degradation

→ I (Lea) recalculated the amount of template needed and I did receive a different outcome: 45 ng/20uL. This is a 15ng difference from what was used in this experiment.

Conclusion: The new samples need to be made in the following experiment.

14th of August 2024

Participants:

• Adrian

Experimental:

Mastermix prepared:

• Tris - 24 ul
• MgCl2 - 15ul
• DTT - 30ul
• Spermidine - 24ul
• rNTPs - 120ul
• Milliq - 387 ul

NOTE: 120ul left after experiment and labelled MM

Samples prepared in following manner added from right to left:

• A: 40 ul mastermix; 1 ul Ribulock RNAse inhibitor;
• B: 40 ul mastermix; 1 ul Ribulock RNAse inhibitor;
• C: 40 ul mastermix; 1 ul Ribulock RNAse inhibitor;
• D: 40 ul mastermix; 1 ul Ribulock RNAse inhibitor;
• E: 40 ul mastermix; 1 ul Ribulock RNAse inhibitor;
• F: 40 ul mastermix; 1 ul Ribulock RNAse inhibitor;
• G: 40 ul mastermix; 1 ul Ribulock RNAse inhibitor;
• H: 40 ul mastermix; 1 ul Ribulock RNAse inhibitor;
• I: 40 ul mastermix; 1 ul Ribulock RNAse inhibitor;
• J: 40 ul mastermix; 1 ul Ribulock RNAse inhibitor;
• K: 40 ul mastermix; 1 ul Ribulock RNAse inhibitor;
• L: 40 ul mastermix; 1 ul Ribulock RNAse inhibitor;
• Stored in freezer (-20C) overnight
• The next day, the T7 poly was added to all samples, and the mRNA in the following manner: A, B - F8 1, C, D - F8 2, E, F - F8 3, G, H - F8 4; I, J, K, L - GFP cDNA (linearized).
• The samples were left for incubation for 4h at 37C, and then left at 4C overnight.

Results and Discussion:

RNA concentration in the samples (ng/ul)

• A - 2937.9
• B - 3080.2
• C - 2760.7
• D - 3565.7
• E - 3172.1
• F - 3296.7
• G - 2965.1
• H - 3077.8
• I - 4896.8
• J - 3059.2
• K - 0.3; 4722.5
• L - 3134.5

Low ratio of A230/A260 indicates contamination of the samples, hence sample purification is needed in the following experiment.

Conclusion: RNA samples show relatively high levels of contamination. Next step is to purify & analyze this RNA.

16th of August 2024

Participants:

• Henriete
• Thijmen

Experimental:

1. 20uL of a GFP mRNA and F8 mRNA samples (A, L, J, H, F, E) was combined with 100uL Phenol/Chloroform/Isoamyl alcohol (https://www.carlroth.com/nl/nl/a-to-z/rotiphenol-chloroform-isoamyl-alcohol/p/a156.1) and 80 ul MilliQ. NOTE: The samples where left on ice before any shaking around XXX minutes due to absence of gloves. samples were shaken vigorously in the fume hood for 15 seconds.
2. centrifuged at 12000 G at 4C for 2 min. NOTE: J was pipetted in F2 (denoted as J2) and F was pipetted in J2 (denoted as F2)
3. another 100 ul of Phenol/Chloroform/Isoamyl alcohol was added to the aqueous samples, shaken and centrifuged for 2 min at 4C 12000G. NOTE: J2 and F2 had no phase separation. they were vortexes again, still nothing.
4. 100 ul of J2 and F2 were taken and the aqueous layers of the other samples. 100 ul of chloroform was added.

Participants:

• Kate
• Thijmen

1. 100 ul of chloroform was added and shaken vigorously
2. samples were shaken vigorously in the fume hood for 15 seconds.
3. centrifuged at 12000 G at 4C for 2 min
4. the aqueous phase was transferred to another epp
5. 10ul of 3 mol sodium acetate was added together with 300 ul ethanol
6. samples were shaken vigorously in the fume hood for 15 seconds.
7. centrifuged at 25000G at 4C for 10 min
8. 50 ul of 70% ethanol was added
9. samples were shaken vigorously in the fume hood for 15 seconds.
10. centrifuged at 25000G at 4C for 5 min
11. 50 ul of 70% ethanol was added
12. samples were shaken vigorously in the fume hood for 15 seconds.
13. centrifuged at 25000G at 4C for 5 min
14. 10 ul of milliQ were added to the samples.

Participants:

• Kate

• Denaturing agarose gel was prepared:
• 0.35 g of agarose gel was added to 36 ml of MilliQ & 5 ml of 10xMOPS.
• It was heated in the microwave for 15 seconds.
• In fume hood, 9ml 36.5% formaldehyde was added to solution while continuously stirring. After that, it was stirred for 5 sec more & added to the casting tray.
• Waited for gel to solidify - 50 min.

20th of August 2024

Participants:

• Henriete
• Thijmen

The samples A-L now are analyzed.

All phases taken were aqueous.

1. 20uL of all GFP mRNA and F8 mRNA samples was combined with 100uL Phenol/Chloroform/Isoamyl alcohol (https://www.carlroth.com/nl/nl/a-to-z/rotiphenol-chloroform-isoamyl-alcohol/p/a156.1) and 80 ul MilliQ.
2. centrifuged at 12000 G at 4C for 2 min
3. C2, A2, I2, G2, D2 and H2 did not have phase separation
4. 100 ul chlorophenol was added to the C1, A1, I1, G1, D1 and H1 and vortexed H1 showed phase separation, A1 did not, C, I, G, D did show. These are supposed to be trash. The “organic layer” from the first step.
5. Another step of chlorophenol was added and samples were centrifuged. Also the trash samples were accidentally centrifuged with chlorophenol only once. The original samples – twice.
6. 100 ul of samples (aqueous layer) were taken from the samples and added 300 ul 100% ethanol.
   • NOTE: Due to labeling inconsistencies, aqueous & organic layers have to be analyzed by adding 10 ul of miliq to all samples - no separation.
   • NOTE: Storage container with all of the materials has been left 15 minutes out of the freezer. (enzymes might go bad).
   • The labels that were labeled similarly initially are labeled with a star (C, D, H, G, J).
7. 10 ul of 3 mol sodium acetate was added & the all were centrifuged at 25000G at 4C for 10 min.
8. The ethanol was taken out.

Experimental:

1. a mastermix for 10 reactions of 20uL was prepared following the protocol for IVT. → ! Guanosine-5’-monophosphate was not used in the reaction mix, because it prohibits capping. mastermix amounts:
   • Tris (1M): 8uL
   • MgCl2 (1M): 5uL
   • DTT(0.1M): 10uL
   • spermidine (25mM): 8uL
   • rNTPs(20mM): 40uL
   • MilliQ: 129uL
   • total: 200uL
2. prepare 10 epps, with each 20uL mastermix
3. 1uL dsDNA of the five different DNA samples (4 FVIII DNA, 1 GFP DNA) was added to epps (for FVIII linearised plasmid, 75 ng of DNA is needed for 20uL reaction, for GFP it is 26ng. The concentration of the FVIII DNA was close enough that 1uL could be taken from the tube. The GFP DNA was diluted 1:1 to a concentration of 30ng/uL and 1uL was used from that )
4. For every different DNA sample, one epp got 2uL T7 polymerase and the other 4uL. → 2uL is the amount recommended by the protocol, but because of the length of the FVIII gene, we wanted to see if a larger amount of T7 would be beneficial.
5. The reaction was incubated at 37 C for 3 hours.
6. 5uL of all samples was mixed with 2uL loading dye and run on an agarose gel (110V 40 min)

Results and Discussion:

Figure 1. Agarose gel electrophoresis of IVT samples. To prevent waste, we use the same agarose gel for multiple experiments. The crossed out lanes are from a previous experiment.

It is weird that 1, 2 and 4 with 2uL T7 did not show a lot of transcription while 3 seems to have a lot, because they should practically be the same. Overall, the reaction seems to work better with 4uL T7. The FVIII RNA was expected to be longer than 7000 instead of 1200. This could be the result of RNA degradation or self splicing, but it could also be because of the properties of the gel and because RNA is not linear. In addition, the GFP RNA did not work out that well in both cases. it could have something to do with the template. maybe the calculations on the amount of template needed was incorrect.

→ I (Lea) recalculated the amount of template needed and I did receive a different outcome: 45 ng/20uL. This is a 15ng difference from what was used in this experiment.

Conclusion: Experiment needs to be repeated with proposed changes:

• different version of T7 poly.
• Add RNAse inhibitor.
• Reduce temperature to prevent autocatalytic breakdown.

Participants:

• Kate

Denaturing agarose gel was prepared:

• 0.35 g of agarose gel was added to 36 ml of Demi water
• It was heated in the microwave for ~ 15 seconds.
• In fume hood, 5 ml of 10xMOPS were added, and then 9ml 36.5% formaldehyde was added to solution while continuously stirring. After that, it was stirred for 5 sec more & added in casting tray.
• Waited for gel to solidify - 50 min.

21st of August 2024

Participants:

• Henriete
• Thijmen

Samples from Friday were noted with 5 at the end of the letter (A, E, H, J, L, F) (ex. A5)

Samples from Tuesday 20.08 were labeled with 2d.

1. Total 13 samples including positive control eGFP
2. 4.5 ul loading dye was added to 1.5 ul of sample.
3. The samples were incubated at 70 degrees for 10 minutes
4. Gel was set to 110 V, 400 A, 20 min (double welled gel)
5. Samples were run in this direction:

• 1st row: F5 A5 L5 H5 E5 J5 eGFP
• 2nd row: B2d L2d J2d F2d K2d E2d

21st of August 2024

Participants:

• Henriete
• Thijmen

Note for agarose gel:

Instead of 0,7 g of agarose, there was 0,82 g of agarose added. This will probably not influence the results but in the future it should be 0,7 g of agarose.

Instead of the normal 0,5x TBE buffer we used in this gel a 1x TBE buffer. This was done by mixing 10 ml of 10x TBE with 90 ml of demi-water.

1. A 1x TBE buffer was made (see notes), to this 0,82 g (see notes) of agarose was added.
2. After this the mixture was swirled and placed in the microwave. Here it was microwaved until the mixture became clear/see through.
3. After this 5 ul of ethidium bromide was added to the mixture after which it was swirled. Now the gel was poured into the tray and the comb was placed in the liquid gel. Now the gel was left to stoll (is this a word?). When the gel was solidified.
4. Gel solidified for 50 minutes.
5. Gel was cut in half.
6. The gel was loaded with a GFP test sample.

Another gel was prepared:

all amounts halved: 45 ml demi H20 + 5 ml TBE 10x. + 2.5 ul EtBr.

This gel was loaded left to right: H5, I2d, F5, K2d, E5, I2d*, J2d*, A5, C2d*, H2d*, B2d, D2d*, F2d. (Figure 6)

23rd of August 2024

Participants:

• Kate
• Thjmen

The new gel was prepared: 100 ml + 0.7 g agarose was heated in the microwave, but the solution boiled, and ~ 25 ml went out. I only took 50 from the solution. 2.5 ul EtBr was added and continuously swirled before added to the tray. The gel was poured in the tray, and cooled for 50 min.

The samples were prepared for loading: 9ul of loading die (2x LB RNA) + 1 ul of sample (14 samples in total) Incubated for 10 min at 70 C.

Results and Discussion:

Labeling: A, B - F8 1, C, D - F8 2, E, F - F8 3, G, H - F8 4; I, J, K, L - GFP

Figure 1. Data lost. Bright - GFP, Dim - FVIII. Most probably: A, E, F, H, J, L

Figure 2. Mutated poly FVIII, wild poly FVIII, GFP test

Figure 1. Loading order: 1st row: F5 (F8) A5 (F8) L5 (eGFP) H5 (F8) E5 (F8) J5 (eGFP) eGFP (test); 2nd row: B2d (F8) L2d (GFP) J2d (GFP) F2d (F8) K2d (GFP) E2d (F8)

Nanodrop:

Sample L and E (showed empty) from Tuesday had errors and also I* and G* had errors. K4 has very low concentration. F2d had very high concentration, but didn’t show on the gel.

Friday (5):

• A5 - 688.9 ng/ul
• E5 - 908.1 ng/ul
• H5 - 2374.7 ng/ul
• J5 - 728.5 ng/ul
• L5 - 2220.9 ng/ul
• F5 - 254 ng/ul

Tuesday:

Figure 2. Concentrations tuesday 1

Figure 3. Concentrations tuesday 2

The nanodrop showed the concentrations of following samples to be faulty: L2d, E2d, I2d* and G2d*. E2d got empty. The analysis was repeated.

They were remeasured with nanodrop: L2d 28.2 ng/ul, others did not give any results (probably, since they had no RNA (the faulty ones due to mislabelling), or due to the presence of ethanol - quick evaporation (probably, ethanol is also present in the L2d).

Figure 4. Masses of the RNAs and their concentrations

The following total masses of RNAs were received. It is weird that F5 has very big concentration, but is not visible on the gel. Same for the B2d, F2d. The gel results are hampered by Ethidium Bromide, and were repeated, but it can be concluded that the samples loaded seem to show the presence of valid compounds, so with that assumption experiments are decided to be continued with these samples.

Figure 5. Test GFP on the cut gel

The new gel made was too thick, so the cutting procedure was done to make it thin, which was successful. The gel made afterwards was made by the techniques described in the background.

Figure 6. This gel was loaded left to right: H5, I2d, F5, K2d, E5, I2d*, J2d*, A5, C2d*, H2d*, B2d, D2d*, F2d.

The GFP mRNA has left the gel, so the gel should be run for less than 50 min (blues line in the middle)

Figure 7. F8 mRNA G2d sample visible, row 10. Visible: 1 (band), 3, kinda 4, 8, 10, 12; Loading order: Frank’s F8; A2d; A5; B2d; C2d; D2d*; E5; F4; F5; G2d; H2d; H5; I2d; Frank GFP

Only F8 G2d was seen, and no GFP - probably ran away.

Conclusion:The FVIII mRNA (G2d) was received in the sufficient amount for further modification - capping and tailing, 7.5 ul, 1428 ng/ul, 10.71 ug.

Next steps:

• Tailing and then capping and analyze with CE
• New IVT of both GFP and FVIII
• Combine all F8 (KEEP G2d PURE!!!) and GFP samples for capping and tailing
• Analyze combined F8 on the gel again

26th of August 2024

Participants:

• Henriete
• Thijmen

IVT was performed by following the following protocol:

1. a mastermix for 10 reactions of 40 ul was prepared by adding the following compounds to an empty epp. mastermix amounts:
   • Tris (1M): 16uL
   • MgCl2 (1M): 10uL
   • DTT(0.1M): 20uL
   • spermidine (25mM): 16uL
   • rNTPs(20mM): 80uL
   • MilliQ: 258uL
   • total: 400uL
2. The master mix was then very shortly vortexed and divided over 10 PCR tubes labelled A to J.
3. After this 1ul RiboLock RNase inhibitor, 1.78 ul sample (F8-1, F8-2, F8-3, F8-4 and GFP) and 8ul polymerase was added to each tube.
4. After this the samples were incubated for 3 hours at 37 degrees and stored in the freezer overnight.

What DNA each tube contains (I have to check the freezer for this):

• A = F8
• B = F8
• C = GFP
• D = F8
• E = F8
• F = F8
• G = GFP
• H = F8
• I = F8
• J = F8

27th of August 2024

Participants:

• Thijmen

This day I wanted to start with purification of the samples from 26-08-2024, but due to phenol oxidation and lack of time, it couldn’t be done. What was done today is as follows:

• 20 ul of sample was pipetted out of the PCR tubes and pipetted into epps labelled A to J.
• To each epp 80 ul of mili Q water was added.

28th of August 2024

Participants:

• Thijmen

This day the purification was performed and analyzed. The purification was done in the following way:

1. 100uL Phenol/Chloroform/Isoamyl alcohol was added to each epp.
2. The epps were shaken vigorously for 15 seconds in the fume hood.
3. The epps were centrifuged for 2 minutes at 12000 rpm at 4C.
4. The aqueous phase was transferred to another epp.
5. 100uL Phenol/Chloroform/Isoamyl alcohol was added to each epp.
6. The epps were shaken vigorously for 15 seconds in the fume hood.
7. The epps were centrifuged for 2 minutes at 12000 rpm at 4C.
8. The aqueous phase was transferred to another epp.
9. 100uL chloroform was added to each epp.
10. The epps were shaken vigorously for 15 seconds in the fume hood.
11. The epps were centrifuged for 2 minutes at 12000 rpm at 4C.
12. The aqueous phase was transferred to another epp.
13. 10 ul of 3 mol sodium acetate was added to each sample
14. 300 ul of 96% (or higher) ethanol was added to each epp.
15. Samples were centrifuged for 10 minutes at 25000 rpm at 4C
16. The ethanol was pipetted out of the epps with care to not disturb the pallet (not every epp had a pallet but most did).
17. 50 ul of 70% ethanol was added.
18. Samples were shaken vigorously in the fume hood for 15 seconds.
19. Centrifuged at 25000G at 4C for 5 min.
20. The ethanol was pipetted out of the epps with care to not disturb the pallet (not every epp had a pallet but most did).
21. 50 ul of 70% ethanol was added to the epps.
22. Samples were shaken vigorously in the fume hood for 15 seconds.
23. Centrifuged at 25000G at 4C for 5 min.
24. The ethanol was pipetted out of the epps with care to not disturb the pallet (not every epp had a pallet but most did).
25. The epps were left to dry for about 20 to 30 minutes.
26. When the epps were dry 10 ul of mili Q water was added to the epps. The epps were labelled as “sample letter + AQ3” for example sample A was labelled as “A AQ3”.

After the purification was done the samples were loaded on a gel which was made the day before. To load the samples the following protocol was used:

1. In each epp 9 ul of loading dye was pipetted and 1 ul of sample.
2. Samples were then lightly vortexed.
3. The epps were then placed in a heating block for around 10 minutes at 70 degrees.
4. After this 9 ul of the mixture was pipetted out of the epp and into the gel. The wells were filled in the following order: A-B-C-D-E-F-G-H-I-J-F8,1 (capped and tailed)-F8,2 (capped and tailed)-GFP (capped and tailed).
5. The gel was then run for 40 minutes at 120 V.
6. After this the gel was placed on a UV machine (don't know the exact name) and was analysed.

Further notes: Sample A probably has a bit less volume because of a pipetting error (shouldn’t be more then 1 ul). During the loading the wells looked a bit weird from sample E onwards, it was like there was an air bubble in there.

Results and Discussion:

As can be seen, the gel looks empty which could have multiple reasons, to be sure what the actual reason is that it looks this way Frank is consulted the next day. Frank said that the gel looks very blue while it should look more purple/pink. The reason why it is blue is probably because the gel doesn’t contain any ethidium bromide.

Conclusion: The gel looks empty because of a lack of ethidium bromide in the gel. For future work it is advised to make a new gel and rerun the samples. During the gel running the samples have also been nano-dropped. For this 1,5 ul of each sample was put in the nanodrop machine.

Results and Discussion:

Purely based on the nanodrop results the samples look very promising, they have high concentrations with a good clean curve and no sign of contamination.

Conclusion: Samples look good in the nanodrop, only the gel will tell us whether the samples are truly good. For future await gel results.

29th of August 2024

Participants:

• Thijmen

By the advice of Frank a new gel was prepared today and also a ‘gel making station’ was setup in lab 3 (from 30-08-24 in lab 4).

The gel was made in the following way:

1. 1L of 1x TBE buffer was made by adding 100 mL of 10X TBE to 900 mL demi-water. This Liter of 1X TBE was used to make the gel and to fill the electrophoresis basket.
2. 0,7g of agarose was weighed and placed in an Erlenmeyer containing 100 mL 1X TBE buffer.
3. This mixture was then heated in the microwave until it was clear and see-through.
4. After this 5 ul of ethidium bromide was added to the mixture.
5. The mixture was poured into the gel tray where is was solidified over a 50 minute period.
6. Next, the gel tray was placed in the electrophoresis basket and the basket was filled with 1X TBE buffer until the gel was submerged.
7. After this the gel was ready to be loaded.
8. This was again done by mixing 9 ul of loading dye with 1 ul of sample.
9. This mixture was then denatured in a heating block for 10 minutes at 70 degrees.
10. After this 9 ul of the mixture was pipetted out of the epp and into the gel. The wells were filled in the following order: A-B-C-D-E-F-G-H-I-J-F8,1 (capped and tailed)-F8,2 (capped and tailed)-GFP (capped and tailed)-Positive sample.
11. The gel was then run for 30 minutes at 120 V.
12. After this, the gel was placed on a UV machine (don't know the exact name) and was analysed.

Results and Discussion:

Most of the samples that were loaded didn’t show up on the gel. We did see a clear band for sample C and the modified GFP. We also see some very vague smears at samples B, D and E. We think that there is again something wrong with the gel because the gel results and the nanodrop results do not match. What is also weird is that our positive sample didn’t show up which could be both a fault in the gel or a fault in the positive sample.

Conclusion: After consulting with Frank, the most likely reason why the gel doesn’t show anything and the nanodrop shows high concentrations is that the mRNA probably is degraded into little mRNA pieces, which would then run out of the 0,7% agarose gel but will still show a high concentration on the nanodrop. The modified GFP looks good and can be used for vesicle experiments!! In the future, it is recommended to rerun the bad samples (so not sample C and modified GFP) on a 2% agarose gel to see whether there is degraded mRNA. Also, the modified GFP can be used in future experiments, the modified F8 can not be used until further notice.

30th of August 2024

Participants:

• Thijmen

Frank advised to make a 2% agarose gel so that’s what was done as follows:

1. 100 mL 1X TBE buffer was made by adding 10 mL of 10X TBE buffer to 90 mL of demi water. This was made in an Erlenmeyer.
2. To this Erlenmeyer 2 g of agarose was added.
3. This mixture was heated in the microwave until it became clear and seethrough
4. After this 5 ul of ethidium bromide was added to the mixture.
5. The mixture was poured into the gel tray where is was solidified over a 50 minute period.
6. Next, the gel tray was placed in the electrophoresis basket.
7. After this the gel was ready to be loaded for the first time.
8. This was again done by mixing 9 ul of loading dye with 1 ul of sample.
9. This mixture was then denatured in a heating block for 10 minutes at 70 degrees.
10. After this 9 ul of the mixture was pipetted out of the epp and into the gel. The wells were filled in the following order: A-F8,1 (capped and tailed).
11. The gel was then run for 10 minutes at 120 V.
12. After this, the gel was placed on a UV machine (don't know the exact name) and was analysed.

Results and Discussion:

If you closely observe the gel you can see that the first loaded gel (sample A) has something in it. This might be the degraded mRNA of which Frank spoke. This is of course not certain but it is an indication.

Conclusion: We decided to load all the bad samples and run them for a longer period of time to see if we could spot more of these possible fragmented pieces of mRNA.

1. After this the gel was ready to be loaded for the second time.
2. This was again done by mixing 9 ul of loading dye with 1 ul of sample.
3. This mixture was then denatured in a heating block for 10 minutes at 70 degrees.
4. After this 9 ul of the mixture was pipetted out of the epp and into the gel. The wells were filled in the following order: B-D-E-F-G-H-I-J-F8,2 (capped and tailed)-Positive sample.
5. The gel was then run for 30 minutes at 120 V.
6. After this, the gel was placed on a UV machine (don't know the exact name) and was analysed.

Results and Discussion:

We can see in the gel that sample G looks very good, it looks bright and pure which is weird given that it didn’t show up like this on the previous gel. Why this is we don’t know and we should consult Frank about this. Furthermore, we can see that samples A, D and J are mainly a big vague smear which could indicate that these samples contain degraded mRNA fragments. Last but not least we see that the positive control sample shows up in this gel as a little pure line. This is good but also weird because why does it show up here and not on the previous gel? This is again something to discuss with Frank.

Conclusion: Sample G could be a good purified sample which can be used for capping and tailing but it must first be discussed with Frank because why didn’t it show up on the previous gel? As for samples A, D and J we can say that these are degraded mRNA fragments but just to check show Frank. Also, ask Frank why the positive sample shows up on this gel but not the previous one. I think these are the best steps to take for the next lab day.

26th of August 2024

Participants:

• Henriete
• Thijmen

Experimental:

1. RNA and MilliQ in a 1.5 ml microfuge tube was added to a final volume of 15.0 µl.
2. Heated at 65°C for 5 minutes.
3. Samples were left on ice for 5 min.
4. the following components were added in the order specified:
   • Denatured RNA (from above): 15.0 µl for F8 , 19.95 ul for GFP
   • 10X Capping buffer: 2.0 µl for F8, 2.66ul for GFP
   • GTP (10 mM) :1.0 µl for F8, 1.33 ul for GFP
   • SAM (2 mM, dilute 32 mM stock to 2 mM): 1.0 µl for F8, 1.33ul for GFP
   • Vaccinia Capping Enzyme: 1.0 µl for F8, 1.33 ul GFP. Incubate at 37°C for 30 minutes.
5. Incubate at 37°C for 30 minutes.
6. RNA is now capped and ready for use in downstream applications.

• NOTE: when adding the GTP, 1.33 ul was accidentally added to all samples. It technically should not change the proficiency of capping as it is the guanosine triphosphate.
• NOTE: The samples were prepared until vaccina enzyme and left on ice for a certain time due to the heating block being too hot and took a while to cool down

Purification of the capped samples:

1. 80 ul MilliQ and 100 ul of chloroform was added and shaken vigorously
2. samples were shaken vigorously in the fume hood for 15 seconds.
3. centrifuged at 12000 G at 4C for 2 min
4. the aqueous phase was transferred to another epp
5. 10ul of 3 mol sodium acetate was added together with 300 ul ethanol
6. samples were shaken vigorously in the fume hood for 15 seconds.
7. centrifuged at 25000G at 4C for 10 min
8. 50 ul of 70% ethanol was added
9. samples were shaken vigorously in the fume hood for 15 seconds.
10. centrifuged at 25000G at 4C for 5 min
11. Pellet was dried and 10 ul MilliQ was added.

Results and Discussion:

As far as we can tell the capping was done successfully since we followed the provided protocol, however, we can’t check right now if it has been successful since we will only place the samples on the gel when the whole RNA modification is done. Tomorrow we will continue with the tailing so for now the samples are stored in the freezer (-20 degrees Celcius).

Conclusion: The RNA capping has been done successfully, so we can proceed with tailing the samples tomorrow.

27th of July 2024

Participants:

• Henriete

GFP sample has 13.3 ug = 1.33 times more than required in protocol, so the volumes of materials for the GFP sample were increased. To make 1X E. coli Poly(A) Polymerase (5X) Reaction Buffer: 4ul E. coli Poly(A) Polymerase + 16 ul water =20 ul 1x E. coli Poly(A) Polymerase Reaction Buffer

Add the following components in the order specified:

1. RNA + MilliQ to a total volume of 15 ul for F8 (19.95 ul for GFP)
2. 5X E. coli Poly(A) Polymerase Reaction Buffer: 2 µl (1X) for F8 (2.66 ul for GFP)
3. ATP (10mM): 2 µl for F8 (2.66 ul for GFP)
4. E. coli Poly(A) Polymerase: 1 µl for F8 (1.33 ul for GFP)
5. Total volume 26.6 ul for GFP and 20 ul for F8
6. Incubate reaction at 37°C for 30 minutes.
7. Stop the reaction by adding EDTA to a final concentration of 10 mM or directly proceeding to the cleanup step.

Participants:

• Henriete

1. 80 ul (73.4 ul for GFP sample) MilliQ and 100 ul of chloroform was added and shaken vigorously
2. samples were shaken vigorously in the fume hood for 15 seconds.
3. centrifuged at 12000 G at 4C for 2 min
4. the aqueous phase was transferred to another epp
5. 10ul of 3 mol sodium acetate was added together with 300 ul ethanol
6. samples were shaken vigorously in the fume hood for 15 seconds.
7. centrifuged at 25000G at 4C for 10 min
8. 50 ul of 70% ethanol was added
9. samples were shaken vigorously in the fume hood for 15 seconds.
10. centrifuged at 25000G at 4C for 5 min
11. Pellet was dried and 10 ul MilliQ was added.

Note: in step 5 only 70% ethanol was added instead of 100%. The 30% water might have dissolved the rna. The samples were centrifuged at 30000G for 5 min again. 100 ul 100% ethanol was added and samples were centrifuged for 5 min at 25000G.

Participants:

• Henriete

1. 100 ml TBE 1x buffer : 90 ml Milliq + 10 ml TBE (10X)
2. 50 ml buffer + 0.35g agarose + 2.5 ul EtBr.
3. Gel was set to solidify for 50 min

Results and Discussion:

Figure 1. gel electrophoresis of modified mRNA (well 11: modified F8 mRNA 1, 12: modified F8 mRNA 2, 13: modified GFP mRNA, 14: positive control) samples to the left belong to RAD131.

Only the modified GFP mRNA is visible on this gel. Suggesting that capping and tailing of this RNA was successful, but that of the F8 RNA was not. This could be because the F8 RNA was already less stable.

Conclusion: The modification was successful on the GFP RNA, but the F8 RNA should be repeated. To get around our problems with purifying the F8 RNA, Frank tried our method.

4th of September 2024

Participants:

• Lea
• Thijmen

Capping:

a new 2 mM SAM dilution was prepared: 2uL (32mM)SAM + 30uL MilliQ. Sample prep:

1. 3.3 uL of sample G was used to get 10ug and diluted with MilliQ to a final volume of 15uL
2. 3.6 ul F8 RNA was used to get 10ug and diluted with MilliQ to a final volume of 15uL
3. Heated at 65C for 5 min
4. placed on ice for 5 min
5. the following components were added in the order specified:
   • 10X Capping buffer: 2.0 µl
   • GTP (10 mM):1.0 µl
   • SAM (2 mM, dilute 32 mM stock to 2 mM): 1.0 µL
   • Vaccinia Capping Enzyme: 1.0 µl.
6. Incubate at 37°C for 30 minutes. (left the sample on ice for a bit while waiting for the heating block to cool down

purification: (same protocol as in RAD129)

1. 80 ul MilliQ and 100 ul of chloroform was added to the sample and shaken vigorously in the fumehood for 15 seconds
2. centrifuged at 12000 G at 4C for 2 min
3. the aqueous phase was transferred to another epp
4. 10ul of 3 mol sodium acetate was added together with 300 ul ethanol. samples were shaken vigorously in the fume hood for 15 seconds.
5. centrifuged at 25000G at 4C for 10 min
6. supernatant was removed and discarded
7. 50 ul of 70% ethanol was added
8. samples were shaken vigorously in the fume hood for 15 seconds.
9. centrifuged at 25000G at 4C for 5 min
10. supernatant was removed and the pellet was dried to the air
11. pellet was resuspended in 15ul milliQ

5th of September 2024

Participants:

• Lea
• Thijmen

tailing: (same protocol as RAD130)

1. reaction mixtures for both samples were prepared by adding the following components:
   • RNA: purified sample
   • 4ul 5X E. coli Poly(A) Polymerase Reaction Buffer
   • 2ul ATP (10mM)
   • 1ul E. coli Poly(A) Polymerase
2. Incubate at 37°C for 30 minutes
3. purification:(same protocol as above)
4. NOTE: a different centrifuge was used with a max RPM of 20000 instead of 25000

6th of September 2024

Participants:

• Maks
• Thijmen

analysis:

The two samples were analyzed on a 0.7% 50 min 110V

Results and Discussion:

Figure 1. After capping and tailing the gel showed no stain

Even though the capping went well (pellet was seen), the tailing went bad (no pellet, even though the pellet should become even bigger). This might be due to absence of Mg2+ in the tailing enzyme.

Conclusion: The capping procedure from the experiment RAD 132 worked out. However, after the tailing procedure no product was seen. The gel showed no stain. The Mg2+ will be added to the tailing enzyme.

11th of September 2024

Participants:

• Kate
• Lea
• Thijmen

Experimental:

• 2 mM SAM dilution was prepared: 2uL (32mM)SAM + 30uL MilliQ
• 8 samples were prepared (4GFP & 4 F8):
• F8: 3.6 ul F8 RNA was used to get 10ug and diluted with MilliQ to a final volume of 15uL (4x)
• GFP(1): 3.3 uL of sample G was used to get 10ug and diluted with MilliQ to a final volume of 15uL
• GFP(4): 6.68 ul GFP from RAD120 1 to get 10ug and diluted with MilliQ to a final volume of 15uL
• GFP(2,3): 5.47 ul GFP from RAD120 2 to get 10ug and diluted with MilliQ to a final volume of 15uL
• NOTE: G sample is empty.

Samples were incubated at 65C for 5 min

Samples were cooled on ice for 5 min

The following components were added in the order specified:

• 10X Capping buffer: 2.0 µl
• GTP (10 mM):1.0 µl
• SAM (2 mM, dilute 32 mM stock to 2 mM): 1.0 µL
• Vaccinia Capping Enzyme: 1.0 µl.

Incubate at 37°C for 30 minutes

Purification: (same protocol as in RAD129)

For each sample:

1. 80 ul MilliQ and 100 ul of chloroform was added to the sample and shaken vigorously in the fumehood for 15 seconds
2. centrifuged at 12000 G at 4C for 2 min
3. the aqueous phase was transferred to another epp
4. 10ul of 3 mol sodium acetate was added together with 300 ul ethanol.
5. centrifuged at 25000G at 4C for 10 min
6. Supernatant was removed and discarded
7. 50 ul of 70% ethanol was added
8. samples were shaken vigorously in the fume hood for 15 seconds.
9. centrifuged at 25000G at 4C for 5 min
10. supernatant was removed and the pellet was dried to the air
11. Pellet was resuspended in 11 uL milliQ
12. Samples were stored at -20C

13th of September 2024

Participants:

• Lea
• Thijmen

Tailing:

For every sample: the following components were added:

• RNA: purified sample in 11 uL milliQ
• 4ul 5X E. coli Poly(A) Polymerase Reaction Buffer
• 2ul 25mM MnCl2 (final conc. Of 2.5 mM)
• 2ul ATP (10mM)
• 1ul E. coli Poly(A) Polymerase
• Final volume: 20uL

NOTE: potential small pipetting error in F8-1 with adding PAP

Incubated at 37C for 30 min.

Purification:

Analysis:

1. 0.7% agarose gel in 1×PBE buffer
2. We recieved 3 samples (2xF8, 1xEGFP) capped and toilet RNA from Frank to also be analysed -> named FF81, FF82 and FGFP
3. Samples were prepaired with 9ul loading dye, 1ul sample, heated to 70C for 20min
4. Samples were loaded in the following order: F81-4, GFP1-4, FF81-2, FGFP.

Results and Discussion:

• 0.7% Agarose gel electrophoresis of denatured mRNA samples:
• 1st, 2nd, 3rd, 9th row shows a smear (F8), 11th row shows a band (GFP form Frank)
• Other lanes are empty
• Reason assumed: concentrations of the sample are too low to be visible.

Nanodrop results:

Conclusion: The concentration of RNA was too low to be seen on the gel, but due to the time constraints, it was decided to move on with this RNA.

9th of July 2024

Participants:

• Adrian
• Kate
• Lea

Experimental:

1. The 5 ml of 1,2-epoxydodecane were added to 25 ml roundbottom flask.
2. The 0.981 ml 1,4-Bis(3-aminopropyl) piperazine were added to the vessel as well. Upon addition, the mixture became whitish-cloudy and then cloudiness has dissappeared.
3. The reaction was heated up to 90C.
4. TLC was conducted on the 1,2-epoxydodecane & 1,4-Bis(3-aminopropyl) piperazine:
   1. 10%MeOH/DCM: 0.5ml/4.5ml + triethyl amine: 0.2ml; Rf(1,2-epoxydodecane) = 0; Rf(1,4-Bis(3-aminopropyl) piperazine) = 1.5/6.3 = 0.24
      
   2. 15%MeOH/DCM: 1.5ml/8.5ml + triethyl amine: 3 drops; Rf(1,2-epoxydodecane) = 0; Rf(1,4-Bis(3-aminopropyl) piperazine) 1/6.8= 0.15
      
5. TLC was conducted on the product & 1,4-Bis(3-aminopropyl) piperazine:
   1. 15%MeOH/DCM: 2ml/11ml + triethyl amine: 5 drops (0.4 ml); Almost no starting material was seen. However, the Rf value was too big. Rf(product) = 4.7/5.1 = 0.92; Rf(1,4-Bis(3-aminopropyl) piperazine) = 0.5/4.4 = 0.113
      
   2. 5%MeOH/DCM: 0.5ml/9.5ml + triethyl amine: 0.3 ml; Two spots were accidentally left for the product next to each other; Rf prod = 1.2 / 3.4 = 0.35; Rf epoxide = >0.95; Rf amine = 0.07;
      
   3. Reaction was left to run for 1 more day. TLC: 5%MeOH/DCM: 0.5ml/9.5ml + triethyl amine: 0.3 ml; TLC showed presence of product and epoxide and no signs of NH2, indicating that the reaction has gone to completion. RF product = 0.38; RF epoxide = 9.25; RF NH2 = 0.09
      
6. The product was left in the freezer at -25C, as yellow crude oil.
   
7. After 2 days, the product was taken out of the freezeer, it became yellowish solidified liquid. The weight of the unpurified compound was measured: 4.224 g.
   
8. The compound of the amount of: 1.018 g was run on the flash column.
9. The following column was prepared: 50 g silica, 3 cm diameter column, 15 cm height silica.
10. Eluent was prepared: 50 ml MeOH/ 950 ml DCM (5%MeOH/DCM), but triethylamine was not added.
11. Fractions 4, 5, 14-26 were stained with annis stain. And fractions 4, 5, 6, 14 - 26 were stained with permanganate stain.
    
12. TLC (5%MeOH/DCM + triethylamine 3 drops) has been done on the fractions: 3, 4, 5, 14, 18, 22, 26.
    
13. New fractions 1*-13* were made.
14. Fractions 14-3* showed the presence of compound, and were combined.
    
15. Fractions 14-3* were rotovapped 900-500 mbar, 55C, ~15min.
16. Samples were submitted for the 1H, 13C, COSY - NMR in CDCl3 and LC-MS.

Results and Discussion:

Yield: crude yellow oil, 1.018 g (pure), 4.224 g (total - not pure), 0.00451 mol, 94.5%; 1H-NMR (300; CDCl3, RT), ppm: 0.9 (A, CH3, m), 1.3 (B, CH2, m), 2.15 (acetone), 2.5 (C, NC, m), 3.5 (D, COH, m), 5.25 (DCM), 7.3 (CDCl3); 13C-NMR (300; CDCl3, RT), ppm: 15 (A, CH3, s), 25-35 (B, CH2, many s), 30 (acetone), 50-60 (C, CN, many s), 60-70 (D, COH, many s), 78 (CDCl3); COSY-NMR (300; CDCl3, RT), ppm: 1 to 1.5, 2.5 to 3.5, 1.5 to 3.5; HSQC-NMR (300; CDCl3, RT), ppm: 3.75 (H) - 70 (C); 2.5 (H) - 60 (C); 1.5 (H) - 25~35 (C); 1(H) - 10 (C).

The results of the analysis show that the 1H-NMR, 13C-NMR, COSY-NMR, and HSQC-NMR have the needed compound. However, COSY-NMR did not show the specific bond found between the B and D region. This was considered a possible mistake in the machine, and the needed structure was confirmed by the HSQC-NMR and LC-MS.

Figure 1. 1H-NMR of ionizable lipids in CDCl3 at RT.

Figure 2. Reference NMR spectra from the [1].

Figure 3. 13C-NMR of ionizable lipids in CDCl3 at RT.

Figure 4. COSY- NMR of ionizable lipids in CDCl3 at RT.

Conclusion: The compound was synthesized correctly, with high purity and high enough yield for the next experiments.

• Kim, M., et al. “Engineered Ionizable Lipid Nanoparticles for Targeted Delivery of RNA Therapeutics into Different Types of Cells in the Liver.” Science Advances, vol. 7, no. 9, 26 Feb. 2021, https://doi.org/10.1126/sciadv.abf4398.
• HYUKJIN, LEE , et al. “Ionizable Lipid Compounds, Lipid Nanoparticles Comprising Same and Therapeutic Uses Thereof.” Worldwide.espacenet.com, 27 Feb. 2024, worldwide.espacenet.com/patent/search/family/074140901/publication/US11911485B2?q=pn%3DUS11911485B2. Accessed 30 June 2024.

26th of August 2024

Participants:

• Adrian
• Kate

Experimental:

• The samples DSPE PEG - 2000, DOPC, & DOPE were dissolved in chloroform, which we have evaporated.
• In 26 minutes, DOPC evaporated under N2.
• In 31 minutes, DSPE PEG - 2000 (PEG) evaporated under N2.
• In around 30 minutes, DOPC evaporated under N2.
• These samples were vacuumed in a desiccator chamber for 15 minutes, and then left overnight to dry.

27th of August 2024

Participants:

• Adrian
• Kate

The next day they were resuspended in 50 ul of ethanol, concentration - 50 mg/ml for DOPC and DOPE, and 10 mg/ml of PEG.

28th of August 2024

Participants:

• Adrian
• Kate

• The samples were nitrogen blown for 15 seconds to remove oxygen traces.
• Then the samples were sonicated for 5 minutes.
• Citrate 10mM was prepared by the procedure listed in the “LNP procedure."

29th of August 2024

Participants:

• Kate

I weighed 1.5 mg and added it to 70 ml of 10 mM, 4 pH citrate solution.

Lipids were sonicated for 5 min at RT.

I added the mixture of lipids 1 ml to 3 ml mRNA in a citrate solution with a syringe. I stirred them vigorously for 10 min. I stored LNP samples, citrate, and cholesterol in RT, the rest of components at -20C.

• The samples DSPE PEG - 2000, DOPC, & DOPE were dissolved in chloroform, which we have evaporated.
• In 26 minutes, DOPC evaporated under N2.
• In 31 minutes, DSPE PEG - 2000 (PEG) evaporated under N2.
• In around 30 minutes, DOPC evaporated under N2.
• These samples were vacuumed in a desiccator chamber for 15 minutes, and then left overnight to dry.

30th of August 2024

Participants:

• Kate

Experimental:

• I added the LNP sample to fill the whole volume of the membrane chamber, and let it centrifuge for 15 minutes, 5000 g, RT. (I through out salts)
• I added 10xPBS in the space which got freed up by the removed salts, and then let it sit in the centrifuge for 30 minutes, 5000 g, RT. (I collected salts)
• Again, I added 10xPBS in the space which got freed up by the removed salts, and then let it sit in the centrifuge for 30 minutes, 5000 g, RT. (I collected salts)
• I did not add PBS and just let the left sample run in the centrifuge for 30 minutes, 5000 g, RT. (No salts were present.)
• LNPs1 were collected.
• I analyzed the samples with a DLS machine

3rd of September 2024

Participants:

• Kate
• Maks

• Solution of purified LNPs1 turned out purple today.
• In two 1.5 ml membranes, in each, around 1 ml of sample was inserted, and then filled up with a 10x PBS buffer.
• Then, the samples were weighed, and inserted into a centrifuge for an hour at 5000 g, RT.
• Retained mixture was dissolved up to 1500 ul & the elution was also collected.

5th of September 2024

Participants:

• Adrian

• Solution of unpurified LNP (2x1.5ml) pipetted into membrane opening of 2 centrifugal ultrafiltration tubes.
• Tubes are filled with a 10x PBS buffer until the volume reaches the level of the bead of the tube.
• Tubes weighed to be within 0.6g of each other and centrifuged for 1 hr at RT at maximum speed (3750 RPM).
• LNPs2 were collected.
• On the next day, solution LNPs2 also turned purple.

Results and Discussion:

Figure 1. DLS. Size measurement of LNPs1 in the Liposome material mode; Dispersant PBS: Average; 3 single measurements.

Figure 2. DLS. Z-potential measurement of LNPs1 in the Liposome material mode; Dispersant PBS: Average; 3 single measurements.

Cholesterol affects the size of the lipids, maybe this is why bigger than 100.

Figure 3. DLS. Size measurement of LNPs1 in the PEG2000 material mode; Dispersant PBS: Average; 3 single measurements.

Figure 4. DLS. Z-potential measurement of LNPs1 in the PEG2000 material mode; Dispersant PBS

The results above show that our LNP size is around the desirable 100 nm. However, it is a bit more, it is assumed that it is due to the ethanol injection methods uncertainty, so this could be improved with microfluidic mixing.

The Z-potential showed weird results. This is assumed to be related to the high acidity of the sample due to its improper purification - always add the sample only to the highest mark in the membrane chamber, and the rest is buffer. This is probably the reason why some ions and salts did not go away.

Solution of purified LNPs1 turned out purple after several days. This is assumed due to a highly acidic environment, which also proves the above point.The LNPs were not sonicated, and this is why they showed bigger size. The purification method was changed, so it is strange that the LNPs still showed high pH. It potentially might be due to not sufficient dialysis steps (only one hour).

Conclusion: LNPs have turned their color to purple, which suggests that either some reaction with LNPs occurred during their storage, or some contamination happened. LNPs were stored in RT, now they will be stored at 4C. LNPs were taken up by syringe during the dialysis analysis. Now, an aliquot will be taken for the analysis, since there is a suspicion that metal from the syringe might have colored the sample.

Always add the sample only to the highest mark in the membrane chamber, and the rest is a buffer. Also, samples should be purified for more than 1 hour. (Potentially, the PBS buffer should be not 10x, but 1x). The Z-potential was most probably affected by purification.

Finally, sonication should always be performed before the DLS measurement.

Advice: always sonicate LNPs before analysis/usage, since they agglomerate

References:

1. Hamatani, Kiyohiro & Eguchi, Hidetaka & Takahashi, Keiko & Koyama, Kazuaki & Mukai, Mayumi & Ito, Reiko & Taga, Masataka & Yasui, Wataru & Nakachi, Kei. (2006). Improved RT-PCR Amplification for Molecular Analyses with Long-term Preserved Formalin-fixed, Paraffin-embedded Tissue Specimens. The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society. 54. 773-80. 10.1369/jhc.5A6859.2006.
2. Jung, Han Na, et al. “Lipid Nanoparticles for Delivery of RNA Therapeutics: Current Status and the Role of in Vivo Imaging.” Theranostics, vol. 12, no. 17, 2022, pp. 7509–7531, https://doi.org/10.7150/thno.77259.
3. “Cell Press: STAR Protocols.” Cell.com, 2024, star-protocols.cell.com/protocols/3495#step-by-step-method-details.

3rd of September 2024

Participants:

• Maks

Experimental:

Compounds were mixed together in the same way as RAD202. NOTE: Yeast mRNA in citrate solution was left out of the fridge overnight.

9th of September 2024

Participants:

• Kate

Figure1. NOTE: Upon storage at -4C, and not interacting with DLS metallic syringe, the LNPs have not turned purplish!

Encapsulation efficiency analysis

Add 0.24 ul of LNPs (stock - 147.5 g/ml) to 10 ml of solvent (PBS 1x) -> 0.0036 g/ml LNPs

Yeast mRNA (was stored at -20C) was warmed to liquid.

The following delusion scheme was prepared: 6 samples dissolve each 2-fold:

11th of July 2024

Participants:

• Kate

DLS analysis

• The LNPs were taken out of the freezer, and then transferred to another vial with glass pipette. NOTE: LNPs were not purple after withdrawal from the fridge.
• LNPs were NOT sonicated before the measurement.
• LNPs were then sonicated, and measured twice in size, and only one Z-potential measurement (B, C measurements).
• LNPs have turned purple after a while after the first measurement

Figure 0. Purple color change observed after DLS. Reason Unknown. Potentially - metal on the needle/ processes that happen within a DLS machine.

Another aliquot of LNPs was taken, sonicated and analyzed on DLS (D).

Results and Discussion:

Figure 1. Figure 1. DLS before sonification. Size measurement of LNPs3 in the PEG2000 material mode; Dispersant PBS

Figure 2. DLS before sonification. Z-potential measurement of LNPs2 in the PEG2000 material mode; Dispersant PBS

Figure 3. DLS after sonication. Size measurement of LNPs2 in the PEG2000 material mode; Dispersant PBS; DLS after sonication (top 3). Z-potential measurement of LNPs2 in the PEG2000 material mode; Dispersant PBS (middle one); DLS after sonication. Size measurement of LNPs2 in the Liposomes material mode; Dispersant PBS (bottom 3)

After DSL LNPs turn purple for unknown reasons. In the 1st measurement RAD203 I did sonicate LNPs (helps agglomerate them), so I measured them again after sonication, and received too big of sizes of LNPs which proved that something changes them after DLS and purple color is the consequence.

Figure 4. Setup of the DLS

The LNPs in the first DLS measurement were not sonicated, which gave results with a bigger size. They were then sonicated after the measurement, and taken for a second DLS measurement.

After the first DLS measurement, the LNPs have turned purplish, and gave weird results in the second DLS measurement.

Encapsulation efficiency

Analysis was not successful, potentially due to the procedure performed in a rush. The background noise value was for some reason showing values bigger than the measured values, which of course can not be the case.

Conclusion: The LNPs hich are measured for the second time after DLS show weird change to purple color, and potentially form conglomerates, since the size values of LNPs in the second DLS increased significantly.

Z potential still behaves weirdly. It is assumed that the following behavior might be due to the low pH in the LNP solution which makes ionizable lipids gain positive charge, so in the next experiment, 1xPBS with higher pH (~7) will be used to check if Z will change. Encapsulation efficiency analysis will be performed more carefully in the following experiments, more time will be provided for the procedure to prevent potential mistakes.

13th of September 2024

Participants:

• Adrian
• Kate

Experimental:

• The LNP beaker was cleaned with ethanol & then acetone.
• Lipids were sonicated for 5 min at RT, and formulated in LNPs with following proportion:

  	Added ul	Theory ul
  ionizable lipids	3.88	3.881271282
  DOPE	2.325	2.325207452
  cholesterol	5.24	5.236352597
  PEG-lipid	3.259	3.258548536
  Ethanol	985	985.2986201

• Dialysis tubes were cleaned after experiment RAD203, LNP, cleaning of dialysis tubes.
• I added the mixture of lipids 1 ml to 3 ml mRNA in a citrate solution with a pasteur pipette dropwise.
• Lipids were mixed together for 10 (+-2) min.
• I have dialysed the LNPs at 3270 g for 1 h in PBS1x, then resuspended them in PBS1x, and set them for dialysis for another hour.
• The LNPs were suspended up to 1.5 ml PBS. Total volume - 3 ml.
• LNP4 were stored at -4C for 3 days.

16th of September 2024

Participants:

• Kate

DLS analysis.

Results and Discussion:

Figure 1. Agarose gel electrophoresis of IVT samples. To prevent waste, we use the same agarose gel for multiple experiments. The crossed out lanes are from a previous experiment.

Figure 2. DLS Z-potential measurement of LNPs204 in the Liposome material mode; Dispersant PBS

In this experiment, LNPs were sonicated before the measurement, so the size of theirs is as expected - around 100 nm [1]. However, the Z-potential was still showing weird numbers - high positive which means that particles are very dispersed in the mixture, but for some reason positively charged. It is assumed that these particles are behaving this way because the LNP composition is different from [1]. PEG-2000 is used, and no mannose PEG is present. It is assumed that the mannose must provide LNPs with some sort of shielding effect, negating their positive charge.

Conclusion: The Z potential seems to be high due to high dispersion of the LNP particles, and high which is a difference from the reference [1] possibly due to different LNP composition. This means that PBSx1 or PBSx10 do not affect the Z, but the properties of particles.

References:

1. https://www.science.org/doi/full/10.1126/sciadv.abf4398?rfr_dat=cr_pub++0pubmed&url_ver=Z39.88-2003&rfr_id=ori%3Arid%3Acrossref.org

16th of July 2024

Participants:

• Kate

Experimental:

Lipids were sonicated for 5 min at RT, and formulated in LNPs with following proportion:

For 5xFV GFP, the stock was diluted by 96.11865483

17th of July 2024

Participants:

• Kate

The following set-up was used:

Hot plate for f8 got heated to 33C for 5 min, then switched off. LNPs were added not while mixing, but mixing started right away. 10 minutes. Then, 7th sample - no-mRNA LNPs were prepared separately, and lipids were added correctly - during the spinning.

18th of July 2024

Participants:

• Kate
• Maks

LNPs were purified with a dialysis machine. 1h, 3290xg, RT 2x times in PBS10x buffer.

18th of July 2024

Participants:

• Kate

TEM

• Sample 3 fell
• Sample 4 carbon film might be worse than for the other grids
• Sample 1: no mRNA - 15 LNPs/view frame: 12000x magnification

19th of July 2024

Participants:

• Kate

DLS was conducted.

Results and Discussion:

It is important to note that the samples with mRNA inside were prepared wrong - lipids were added BEFORE the mixing, and fast, and not during the mixing. This might affect the results of the mRNA LNP complexes.

Figure 1. DLS size measurement of LNPs205 LNPs with no mRNA in the Liposome material mode; Dispersant PBS

Figure 2. DLS Z-potential measurement of LNPs205 LNPs with no mRNA in the Liposome material mode; Dispersant PBS

Figure 3. DLS size measurement of LNPs205 LNPs with GFP control mRNA in the Liposome material mode; Dispersant PBS

Figure 4. DLS Z-potential measurement of LNPs205 LNPs with GFP control mRNA in the Liposome material mode; Dispersant PBS

In the figures above, you can see that the size of the LNPs is off. Both, for LNPs with no mRNA and with mRNA inside. Even though, when the mixing was done correctly for the LNPs with no mRNA, the size improved, but is still bigger, since no sonification was performed (the concentration of lipids for the no-mRNA was high).

This could indicate that upon injection of ethanol, the ethanol and aqueous phase mix, and do not provide for the right mixing between mRNA and lipids, leading to lipid clusters suspended in the liquid. This is observed only now, and not in previous experiments, since the lipid concentration now is very low, and this affects the method results a lot. It is recommended to use the pulsification method or microfluidics device to prevent this in the future.

TEM

Figure 1. LNPs with no mRNA under TEM

Figure 2. LNPs with mRNAs under TEM - none present

Conclusion: It is crucial to add the lipids DURING the mixing step, and not before, since it prevents from forming LNP-mRNA complex. LNPs almost do not form with the ethanol injection method at very low concentration. It is recommended to use higher concentration of lipids, and implement a pulsification method or microfluidics device to prevent this in the future. The TEM does not show any LNPs with mRNA, but shows the LNPs without. It also shows that encapsulation is complex, and might lead to no results if some aspect is not done correctly (in this case - mixing, and low concentration).

• different version of T7 poly.
• Add RNAse inhibitor.
• Reduce temperature to prevent autocatalytic breakdown.

19th of September 2024

Participants:

• Lea

Experimental:

• transfection solution was prepared:
• 50 ul rNA buffer containing 400ng RNA was mixed with 1ul reagent, mixed and left for 15 min:

  	mRNA iGEM	3CB mRNA	Mock
  RNA	0.8 ul	4ul	-
  Buffer	49.2ul	46ul	50ul
  Reagent	1ul	1ul	1ul

• 3CB is the batch number of the EGFP mRNA given to us from the lab that is known to be functional. This is our positive control.
• 25 ul of transfection mix was added to one well containing HEK cells with +- 50% confluency, 2 wells per sample.
• Cells were incubated with the transfection mix for +- 19h

Results and Discussion:

The 3CB control shows visible expression indicating transfection was successful. However, the expression of the control was expected to be higher. Low expression can be explained by the fact that the cells were not very healthy when the transfection was performed. The expression of our synthesized mRNA is a lot lower, but some cells expressing GFP can be found - like the one indicated by the red arrow. The negative control is empty.

Conclusion:We produced functional mRNA. However, the expression is not optimal. From this we can conclude that our method for synthesizing mRNA works, but could be improved. Due to the low expression of our mRNA, it might be beneficial to use the 3CB GFP mRNA to test the efficiency of the LNPs.

24th of September 2024

Participants:

• Vasilis

1. A dilution of 25 ul was made using 23ul of mRNA buffer
2. 0.5 ul of transfection reagent was added to every sample
3. The samples were incubated for 15 min
4. The cells were labeled with the same numbers as the samples
5. 1ul of each sample was added to each well with the cells

Results and Discussion:

Figure 1. The control GFP mRNA is expressed

Figure 2. Our mRNA samples and LNPs (also the one with control GFP) with mRNA, no GFP detected (same picture for all)

Figure 3. Trans picture of cells with the LNPs inside (same picture for all)

Figure 4. Cells without fluorescence - survived

Only the sample with control GFP was expressed. This implies that the mRNA samples are nonfunctional (GFP 1, which was slightly expressed in RAD301, was not visible here, potentially due to denaturation). Since control was expressed by regular transfection, and not with LNPs, the LNP synthesis procedure in the moment is not efficient.

SDS-PAGE gel was considered to be unnecessary for analysis of F8 protein presence, since if there was no expression of control GFP, than yes, but since there is no expression of the experiment with GFP whose RNA also looked better from the start, I think it is a waste of time and resources. In the final effort, I would put my money on getting the transfection both with JetMessenger ánd LNPs to work before analyzing protein extracts of cells.

Conclusion:Combining the results from the RAD206, RAD133, and RAD203, it can be said that the reason for the following transcription result is a combination of the results from all 3 factors: HEK cells are used instead of the cells used in the reference [1] - HeLa or LSEC cells, different lipid composition was used: instead of DSPE-PEG 2000, C16-PEG ceramide (PEG-lipid) and DSPE-PEG-mannose lipids were used, and the mRNA experiments provided very low yield of functionable mRNA.

This resulted in no transfection fluorescence of just GFP mRNA samples (except for the control one), since the mRNA from RAD133 also was not visible on the gel, and in no transfection fluorescence of GFP in LNPs. However, the LNP procedure was also not efficient - very little amount of LNPs produced in combination with different compounds used for production.

It is advised to make more mRNA samples after consultation on the current results, procedure and storage should be revised. Different cell cultures should be used: either HeLa or LSEC cell lines. Also, for LNPs, it is recommended to use higher concentration of lipids, and implement a pulsification method or microfluidics device to prevent low yields of LNPs in the future. It is also recommended to purchase more expensive compounds for LNP formulation, as in the referenced article [1]: instead of DSPE-PEG 2000 - C16-PEG ceramide (PEG-lipid) and DSPE-PEG-mannose lipid which might provide for the better targeting during transfection.

References:

1. https://www.science.org/doi/full/10.1126/sciadv.abf4398?rfr_dat=cr_pub++0pubmed&url_ver=Z39.88-2003&rfr_id=ori%3Arid%3Acrossref.org