Experiment

Table of contents

Molecular Biology Lab


Primer Resuspension

  1. Multiply oligo yield information (in nmol) on the tube label or in the "Amount of oligo" section of your specification sheet, and multiply by 10.
  2. The resulting product is the amount of buffer needed (TE or nuclease-free water), in μL, to prepare a 100 μM solution. For example, if yield is 9 nmol, 90 μL of buffer is needed to make a 100μM solution.
  3. If difficult to dissolve, incubate in a water bath at 55°C for 1 minute.

gBlock Resuspenion

  1. Before opening the tube, spin it down in a microcentrifuge for 3-5 seconds to ensure the DNA is in the bottom of the tube. The pellet can become statically charged and, without this step, can either fly out of the tube or remain in the cap, resulting in loss of yield.
  2. Add molecular grade water, or a buffer such as IDTE, to reach a final concentration of 10 ng/μL. Storage concentrations <1 ng/μL may result in loss of material due to adherence to the plastic tube. The addition of a carrier such as tRNA or poly A at a concentration of 0.1 to 1.0 mg/mL can help to avoid this issue. (Please note that this concentration is just a suggestion, it is acceptable to use a higher concentration if your application requires.)
  3. Vortex briefly.
  4. Incubate at approximately 50°C for 15-20 min. Heating the tube will ensure the solvent comes in contact with the tiny pellet, even if it is stuck to the side of the tube. Thus, this step will increase the likelihood that the entire pellet will be resuspended.
  5. Briefly vortex and centrifuge.

Touchdown PCR

(This is only a template to start from, everything except Q5 volume and total reaction volume can be adjusted depending on results.)
NEB Q5 Hi-Fi 2X Master Mix1 10µL
Forward Primer (100µM) 0.4µL
Reverse Primer (100µM) 0.4µL
Template DNA 4ng
DMSO2 (if needed) 2µL (10% of final volume, decreases Tm by 6°C)
Nuclease-free Water Top up to 20µL
Total Reaction Volume 20µL
Tm Calculate with NEB Tm calculator with the overhang sequence of both primers
Cycles 10 cycles of touchdown PCR, 20-25 cycles of conventional PCR
Extension time 20-30 seconds per kb; for size >9kb, use 40 seconds per kb

Notes:

  1. NEB Q5 Hi-Fi 2X Master Mix and NEB Q5 Hotstart Hi-Fi 2X Master Mix was used interchangeably, with no significant difference in efficiency or quality. Protocols for either Master Mix are the same.
  2. DMSO was only used when Tm was above 72°C. 10% DMSO decreases Tm by around 6°C, and was used to optimize product formation (Chester and Marshak, 1993).

Colony PCR

  1. Prepare an appropriate volume of master mix containing 200nM forward and reverse primers, and nuclease-free water based on the number of colony PCR aiming to perform. For each colony, add 0.4uL of forward and 0.4uL of reverse primer, and 9.2uL of nuclease-free water.
  2. Select the target colonies from the plates and mark them.
  3. With the help of 10uL pipette tip, carefully pick up half of the colony and scratch it on the bottom of the PCR tube.
  4. Add 10uL of the primer mix to each PCR tubes, add 10uL NEB Q5 Hi-Fi 2X Master Mix next.
  5. Perform agarose gel electrophoresis after the PCR is completed.

Agarose Gel Electrophoresis

Expected Fragment Size Agarose Gel Concentration Voltage
>10000bp 0.8% 80 - 160 V (balance speed and not running DNA out of the gel)
100-10000bp 1% 80 - 160 V (balance speed and not running DNA out of the gel)
<100bp 3% 80 - 160 V (balance speed and not running DNA out of the gel)
  1. Measure out agarose and TAE needed. With ≤8 lanes to load, 25mL of TAE and 0.25g of agarose would be measured for a 1% gel. Add both into a conical flask.
  2. Microwave the mixture for 5s at a time, swirling the flask between microwaving to dissolve the agarose. Repeat until the mixture turns completely clear.
    1. a. Be careful not to boil the mixture.
  3. Let the mixture cool down to around 45°C, when it doesn't hurt to the touch. Add gel stain.
    1. a. GelRed and SYBR Safe at 10,000X concentration was used for this project. In a 25mL gel, 2.5μL of gel stain is added.
  4. Wash gel cast and gel comb with nuclease-free water, then pour mixture into cast and cover with aluminum foil. Rest until solidifies.
  5. Add gel loading dye to samples.
    1. a. NEB blue and purple loading dyes (6X) were used for this project. 2μL of dye were added for 10μL of sample.
  6. Load DNA samples and DNA Ladder into the gel.
    1. a. 8μL of ladder per well and 10μL of sample per well was found to be optimal.
    2. b. If there were excess wells, wells on the edges of the gel were not used since the gel could set unevenly.
  7. Run the gel in the TAE buffer at 120V, until the gel loading dye reaches the midpoint of the agarose gel. Remove and visualize in a gel doc.

Gel Extraction (with Geneaid GenepHlow Gel/PCR Kit)

(Steps labeled with * are modifications to the kit's protocol, which we found to help yield.)

  1. Gel Dissociation
    1. a. Excise the agarose gel slice containing relevant DNA fragments and remove any extra agarose to minimize the size of the gel slice. Transfer up to 300 mg of the gel slice to a 1.5ml microcentrifuge tube. Add 500 μl of Gel/PCR Buffer and mix by vortex. Incubate at 55-60°C for 10-15 minutes to ensure the gel slice has been completely dissolved. During incubation, invert the tube every 2-3 minutes. Cool the dissolved sample to room temperature.
  2. DNA Binding
    1. a. Place a DFH Column in a 2ml Collection Tube. Transfer 800μl of the sample mixture to the DFH Column. Centrifuge at 16,000 x g for 30 seconds. Discard the flow-through and place the DFH Column back in the 2 ml Collection Tube. If the sample mixture is more than 800μl, repeat the DNA Binding step.
  3. Wash
    1. a. Add 400 μl of W1 Buffer into the DFH Column. Centrifuge at 16,000 x g for 30 seconds then discard the flow-through. Place the DFH Column back in the 2ml Collection Tube. Add 600 μl of Wash Buffer into the DFH Column. Let stand for 1 minute at room temperature. Centrifuge at 16,000 x g for 30 seconds then discard the flow-through. Place the DFH Column back in the 2 ml Collection Tube. Centrifuge at 16,000 x g for 3 minutes to dry the column matrix.
    2. b. Repeat step 3a once.*
    3. c. Centrifuge at 16,000 x g for 2 minutes to dry the column matrix.*
    4. d. Open the cap of the column and leave it on the benchtop to dry for 2 minutes.*
  4. DNA Elution
    1. a. Transfer the dried DFH Column to a new 1.5ml microcentrifuge tube. Add 20μl of (60) pre-heated Elution Buffer into the center of the column matrix. Let stand for at least 2 minutes to ensure the Elution Buffer is completely absorbed. Centrifuge for 2 minutes at 16,000 x g to elute the purified DNA.

PCR Cleanup (with Geneaid GenepHlow Gel/PCR Kit)

(Steps labeled with * are modifications to the kit's protocol, which we found to help yield.)

  1. Sample Preparation
    1. a. Transfer up to 100μl of reaction product to a 1.5 microcentrifuge tube. If the sample is less than 50μl, adjust the volume to 50 μl with ddH2O. Add 5 volumes of Gel/PCR Buffer to 1 volume of the sample and mix by vortex.
  2. DNA Binding
    1. a. Place a DFH Column in a 2ml Collection Tube. Transfer the sample mixture to the DFH Column. Centrifuge at 16,000 x g for 30 seconds. Discard the flow-through. Place the DFH Column back in the 2ml Collection Tube.
  3. Wash
    1. a. Add 600 μl of Wash Buffer (make sure ethanol was added) into the center of the DFH Column. Let stand for 1 minute at room temperature. Centrifuge at 16,000 x g for 30 seconds. Discard the flow-through and place the DFH Column back in the 2ml Collection Tube. Centrifuge for 3 minutes at 16,000 x g to dry the column.
    2. b. Repeat step 3a.*
  4. DNA Elution
    1. a. Transfer the dried DFH Column to a new 1.5 ml microcentrifuge tube. Add 20μl of (55°C) pre-heated Elution Buffer or TE into the center of the column matrix. Let stand for at least 2 minutes to ensure the Elution Buffer is completely absorbed. Centrifuge for 2 minutes at 16,000 x g to elute the purified DNA.

NEBuilder Assembly

  1. Input the length (bp) and concentration (ng/uL) of each fragment at https://nebuildercalculator.neb.com/ to generate the recipe for assembly.
  2. Prepare the reaction on ice.
  3. Incubate at 50°C for 1 hour in a thermocycler. Extend the incubation time to 2 hours for complex assembly with more than 4 fragments.
  4. Take out 3 uL from the ligation mix, and run it in a gel with SDS-loading dye (after 15 min incubation at 65°C) for size verification. Store the rest of the ligation mix in -20°C.
  5. Proceed to chemical transformation.

Chemical Transformation


Prepare LB plates

  1. Add 32 g of LB agar (Lennox) to 1 L of MilliQ water.
  2. Formulation per one liter:
    1. a. 10 g SELECT Peptone 140
    2. b. 5 g SELECT Yeast Extract
    3. c. 5 g Sodium Chloride
    4. d. 12 g SELECT Agar
  3. Autoclave at 121°C for 19 minutes.
  4. Prepare a 15 mL or 50 mL falcon tube.
  5. Pour 15 mL of LB agar to the tube.
  6. Add 150 uL of 100X ampicillin to the tube, invert the tube a few times.
  7. Pour the LB agar to the plate.
  8. Prewarming the plates to 37°C before plating is recommended but not necessary.

Thermofisher DH10B competent cells transformation

  1. Put S.O.C medium in room temperature.1)
  2. Thaw competent cells on wet ice.
  3. Place the required number of 1.5-mL polypropylene microcentrifuge tubes on wet ice.
  4. Gently mix the cells, then make 50 µL aliquots of competent cells in the chilled 1.5-mL microcentrifuge tubes.
  5. Add 1-5 µL of sample DNA directly into a tube of competent cells.2)
  6. Mix well by gently flicking the tube several times (do NOTpipette up and down).
  7. Incubate the cells on ice for 30 minutes.
  8. Heat-shock the cells for exactly 30 seconds in a 42°C water bath or incubator.
  9. Do not mix or shake the tube.
  10. Incubate the cells on ice for 2 minutes.
  11. Add 250 µL of room-temperature S.O.C. Medium.
  12. Place the tube on its side in a shaking incubator. Use tape to secure the tube in place.
  13. Shake the tube at 225 rpm for more than 1 hour at 37°C.3)
  14. If necessary, dilute the cells 1:10 with S.O.C. Medium.
  15. Spread at least two different volumes (20-200 µL) of cells from each transformation reaction on separate LB plates containing the appropriate selective antibiotic.4)
  16. Label the plates with the plating volume so that the amount providing the best colony density can be identified.
  17. Invert the plates and incubate overnight at 37°C.5)
  18. Select colonies and analyze by plasmid isolation, PCR, or sequencing.6)


Notes:

  1. S.O.C usually stored in 4°C, it can also be stored at room temperature.
  2. It is better to try different volumes for assembly products. Due to our experience, 1uL or 2uL can reach the highest efficiency for around 0.2 pmol assembly products. Too much volume will also increase the enzyme transform to the cells, which might cause low transformation efficiency.
  3. For large size plasmid, it is better to extend the recovery time. Due to our experience, 2 hour recovery time is appropriate for 15 kb plasmids.
  4. For 15 mL LB plates, up to 300 uL of cells can be spread.
  5. Colonies can be seen after 12 hours, we usually miniculture the colonies after 16 hours. The plates can also be stored in 4°C plates and miniculture when needed.
  6. Colony PCR is also a good way to save miniprep resources. Please refer to the colony PCR protocol.

Invitrogen One Shot TOP10 Chemically Competent E.Coli

  1. Put S.O.C medium in room temperature.1)
  2. Thaw, on ice, one vial of One Shot™ TOP10 chemically competent cells for each transformation.
  3. Add 1 to 5 μL of the DNA (10 pg to 100 ng) into a vial of One ShotTM cells and mix gently. Do not mix by pipetting up and down. For the pUC19 control, add 10 pg (1 μL) of DNA into a separate vial of One Shot™ cells and mix gently.2)
  4. Incubate the vial(s) on ice for 30 minutes.
  5. Heat-shock the cells for 30 seconds at 42°C without shaking.
  6. Remove the vial(s) from the 42°C bath and place them on ice for 2 minutes.
  7. Aseptically add 250 μL of pre-warmed S.O.C. Medium to each vial.
  8. Cap the vial(s) tightly and shake horizontally at 37°C for more than 1 hour at 225 rpm in a shaking incubator.3)
  9. Spread 20-200 μL from each transformation on a pre-warmed selective plate and incubate overnight at 37°C. We recommend that you plate two different volumes to ensure that at least one plate will have well-spaced colonies.4)
  10. For the pUC19 control, dilute the transformation mix 1:10 into LB Medium (e.g., remove 100 μL of the transformation mix and add to 900 μL of LB Medium) and plate 25-300 μL.
  11. Store the remaining transformation mix at 4°C. Additional cells may be plated out the next day, if desired.
  12. Invert the selective plate(s) and incubate at 37°C overnight.5)
  13. Select colonies and analyze by plasmid isolation, PCR, or sequencing.6)


Notes:

  1. S.O.C usually stored in 4°C, it can also be stored at room temperature.
  2. It is better to try different volumes for assembly products. Due to our experience, 1 μL or 2 μL can reach the highest efficiency for around 0.2 pmol assembly products. Too much volume will also increase the enzyme transform to the cells, which might cause low transformation efficiency.
  3. For large size plasmid, it is better to extend the recovery time. Due to our experience, 2 hour recovery time is appropriate for 15 kb plasmids.
  4. For 15 mL LB plates, up to 300 μL of cells can be spread.
  5. Colonies can be seen after 12 hours; we usually miniculture the colonies after 16 hours. The plates can also be stored in 4°C plates and miniculture when needed.
  6. Colony PCR is also a good way to save miniprep resources. Please refer to the colony PCR protocol.

NEB 10-beta Competent E.Coli

  1. Put S.O.C medium in room temperature.
  2. Thaw a tube of NEB 10-beta Competent E. coli cells on ice until the last ice crystals disappear. Mix gently and carefully pipette 50 μl of cells into a transformation tube on ice.
  3. Add 1-5 μL containing 1 pg-100 ng of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex.1)
  4. Place the mixture on ice for 30 minutes. Do not mix.2)
  5. Heat shock at exactly 42°C for exactly 30 seconds. Do not mix.
  6. Place on ice for 5 minutes. Do not mix.
  7. Pipette 950 μL of room temperature NEB 10-beta/Stable Outgrowth Medium into the mixture.
  8. Place at 37°C for more than 60 minutes. Shake vigorously (250 rpm) or rotate.3)
  9. Warm selection plates to 37°C.
  10. Mix the cells thoroughly by flicking the tube and inverting, then perform several 10-fold serial dilutions in NEB 10-beta/Stable Outgrowth Medium.4)
  11. Spread 50-300 μL of each dilution onto a selection plate and incubate overnight at 37°C. Alternatively, incubate at 30°C for 24-36 hours or 25°C for 48 hours.5)
  12. Select colonies and analyze by plasmid isolation, PCR, or sequencing.6


Notes:

  1. It is better to try different volumes for assembly products. Due to our experience, 1 μL or 2 μL can reach the highest efficiency for around 0.2 pmol assembly products. Too much volume will also increase the enzyme transform to the cells, which might cause low transformation efficiency.
  2. For maximum transformation efficiency, cells and DNA should be incubated together on ice for 30 minutes. Expect a 2-fold loss in transformation efficiency for every 10 minutes this step is shortened.
  3. For large size plasmid, it is better to extend the recovery time. Due to our experience, 2 hour recovery time is appropriate for 15 kb plasmids.
  4. For 15 mL LB plates, up to 300 μL of cells can be spread.
  5. Colonies can be seen after 12 hours, we usually miniculture the colonies after 16 hours. The plates can also be stored in 4°C plates and miniculture when needed.
  6. Colony PCR is also a good way to save miniprep resources. Please refer to the colony PCR protocol.

Liquid subculture


  1. Prepare the LB Broth (Miller's)

    2. a. Add 25 g of LB Broth(Miller's) to 1 L of MilliQ water.
    b. Formulation per one liter:
    c. Autoclaved at 121°C for 19 minutes.
    d. Prepare a 15 mL falcon tube.
    e. Pour 5 mL of LB agar to the tube.
    f. Add 50 uL of 100X ampicillin to the tube, invert the tube a few times.

  2. Prepare the subculture

    3. a. Light the alcohol lamp.
    b. Select the target colonies from the plates and mark them.
    c. Use pipette tip to collect the colonies, eject the tip into the tube with 5 mL LB Broth (Miller's).
    d. Loosen the cap of the tube in order to let oxygen in.
    e. Place at 37°C for about 16 hours. Shake vigorously (250 rpm) or rotate.1)

    1) The incubation time is up to 18 hours, more than 18 hours might cause overgrowth.


Miniprep

For the NEB Monarch Plasmid DNA Miniprep Kit, the manufacturer's protocol was followed without modifications.


For the Tiangen TIANprep Mini Plasmid Kit, the manufacturer's protocol was followed without modifications.


IVT

The NEB HiScribe T7 High Yield RNA Synthesis Kit was used, following the "Capped RNA Synthesis" and "Poly(A) Tailing of RNA using E. coli Poly(A) Polymerase" protocols without modifications.


RNA Cleanup

The NEB Monarch RNA Cleanup Kit was used, following NEB's protocol with no modifications.

Cell Lab


Culture

See Thermofisher's “Useful Numbers for Cell Culture” for useful numbers.

  1. Recipe of culture medium

    1. a. RPMI for THP1
    2. b. M10 for HepG2(GFP)
    3. c. DMEM for HepG2(GFP)

  2. Thaw cells from -80°C and liquid Nitrogen

    1. Thaw the cells in a 37°C water bath for 1 minute.
    2. Transfer cells to a Class II biosafety cabinet.
    3. Label 2*15mL tubes with the cell name.
    4. Add 1mL medium to the empty tube.
    5. Transfer all thawed cells into the empty tubes.
    6. Centrifuge at 12x100 RPM for 3 min.
    7. Remove as much supernatant as possible (DMSO, toxic to cells) without disturbing the pellet.
    8. Add 2 mL per plate (for 6-well plate) of medium to the tube and pipette up and down to resuspend the cells.
    9. Take a 6-well plate and label the lid above the well: name, date, passage no. E.g., THP-1 19/6 P3.
    10. Pipette 2mL of the cells into 1 well (6-well plate).
    11. Shake the well in forward-backward and right-left directions 20 times per direction to avoid the clumping.
    12. Incubate the 6-well plate at 37°C 5% CO2.
    13. Check the confluence every day and passage if necessary (see below for protocol).

  3. Cell passage (1:X passage)

    1. 3.1 for THP1

      1. Prepare and label a falcon tube.
      2. Transfer all 2mL of the cells into the tube.
      3. Centrifugation at 12x100 RPM for 3 min.
      4. Remove as much supernatant as possible without disturbing the pellet.
      5. Add 1 mL/plate (for 6-well plate) of medium to the tube and pipette up and down to resuspend the cells.
      6. Transfer 1000/X µL of the cells to the new well.
      7. Add (2000-1000/X) µL of the RPMI (FBS and antibiotics added) to the well, pipette up and down roughly.
      8. Shake the well in forward-backward and right-left directions 20 times per direction to avoid the clumping.
      9. Incubate the 6-well plate at 37°C 5% CO2.
      10. Check the confluence every day and passage if necessary.

    2. 3.2 for HepG2(GFP)

      1. Discard all the medium in the well.
      2. Use 1 mL of PBS to wash the cells 3 times.
      3. Trypsinize HepG2 cells with TrypLE dissociation reagent for 5 min in a 37°C incubator.
      4. Add 1 mL of DMEM (or M10, FBS and antibiotics added) to stop the trypsinizing.
      5. Prepare a falcon tube and label the cell name on it.
      6. Transfer all 2mL of the cells into the tube.
      7. Centrifugation at 12x100 RPM for 3 min.
      8. Remove as much supernatant as possible without disturbing the pellet.
      9. Add 1 mL/plate (for 6-well plate) of medium to the tube and pipette up and down to resuspend the cells.
      10. Transfer 1000/X µL of the cells to the new well.
      11. Add (2000-1000/X) µL of the DMEM (or M10, FBS and antibiotics added) to the well, pipette up and down roughly.
      12. Shake the well in forward-backward and right-left directions 20 times per direction to avoid the clumping.
      13. Incubate the 6-well plate at 37°C 5% CO2.
      14. Check the confluence every day and passage if necessary.

Electroporation

  1. Thaw THP-1 cells at least 7 days prior and passage two times before plating for transfection.
  2. Cultivate the required number of cells such that the cells are 70-90% confluent on the day of the experiment.
  3. On the day of the experiment, harvest and wash the cells in phosphate buffered saline (PBS) without Ca2+ and Mg2+:

    a. Washing method for THP1:

    i. Centrifuge at 1200g for 3 minutes

    ii. Remove the supernatant, resuspend the pellet with PBS

    iii. Centrifuge at 1200g for 3 minutes

  4. Resuspend the cell pellet in Resuspension Buffer R (included with NeonTM Kits) at a final concentration of 2.0 x 107 cells/mL.
  5. Prepare enough cells for at least five transfections (i.e., 1.0 x 106 cells in 50 µL).
  6. Transfer the cells to a sterile, 1.5 mL microcentrifuge tube.
  7. Prepare 24-well plates by filling the appropriate number of wells with 0.5 mL of RPMI Complete Growth Media without antibiotics and pre-incubate the plates at 37°C in a humidified 5% CO2 incubator. Adjust the volume accordingly if using other plate formats.
  8. Turn on the NeonTM unit and enter the electroporation parameters in the Input window

    a. Parameter:

    i. Voltage (V): 1700 Pulse

    ii. Width (ms): 20

    iii. Pulse Number: 1

    iv. Cells/ml: 2X107

    v. Pulse Number: 1

  9. Fill the NeonTM Tube with 3 mL Electrolytic Buffer (use Buffer E for the 10 µL NeonTM Tip and Buffer E2 for the 100 µL NeonTM Tip).
  10. Insert the NeonTM Tube into the NeonTM Pipette Station until you hear a click sound.
  11. Transfer 1.0 µg plasmid DNA per transfection reaction (i.e., 5.0 µg for 5 transfections) to the tube containing cells and mix gently.
  12. Insert a NeonTM Tip into the NeonTM Pipette.
  13. Press the push-button on the NeonTM Pipette to the first stop and immerse the NeonTM Tip into the cell-DNA mixture. Slowly release the push-button on the pipette to aspirate the cell-DNA mixture into the NeonTM Tip.
  14. Insert the NeonTM Pipette with the sample vertically into the NeonTM Tube placed in the NeonTM Pipette Station until you hear a click sound.
  15. Ensure that you have entered the appropriate electroporation parameters and press Start on the NeonTM touchscreen to deliver the electric pulse.
  16. The touchscreen displays “Complete” to indicate that electroporation is complete.
  17. Remove the NeonTM Pipette from the NeonTM Pipette Station and immediately transfer the samples from the NeonTM Tip into the prepared culture plate containing pre-warmed RPMI complete growth medium without antibiotics.
  18. Discard the NeonTM Tip into an appropriate biological hazardous waste container.
  19. Gently rock the plate to assure even distribution of the cells. Incubate the plate at 37°C in a humidified CO2 incubator.
  20. For the first passage after electroporation, dead cells can be removed:
    1. Prepare a falcon tube and label the cell name on it
    2. Transfer all 0.5mL of the cells into the tube
    3. Centrifugation at 100 RPM for 10 min
    4. Discard the supernatant, remove as much as possible but must not disturb pellet
    5. Add 0.5mL of medium to the tube and pipette up and down to resuspend the cells
    6. Transfer the cells to the new well
    7. Shake the well in forward-backward and right-left directions 20 times per direction to avoid the clumping
    8. Incubate the 24-well plate at 37°C 5%CO2
    9. Check the confluency everyday and decide whether to do the passage

Lipofection (w/ Lipofectamine 2000 for 24-well plate)

  1. Passage the cells into plate with antibiotic free medium (raw RPMI + 15% FBS for THP1)
  2. Mix 0.6uL/0.75uL/2uL of MessengerMAX with 25uL of Opti-MEM medium, incubate for 5 minutes
  3. Dilute 0.6ug/1ug/1ug RNA respectively with 50uL of Opti-MEM medium
  4. Add 25uL of diluted RNA to 25uL diluted MessengerMAX, incubate for 10 minutes
  5. Add the mRNA-lipid complex (50uL) to cells

Please also refer to Thermofisher's “Lipofectamine 2000 Reagent Protocol 2013”


M0 THP-1 Differentiation

  1. Add 2uL 1000X PMA to 2mL 70% to 90% THP1.
  2. Incubate at 37°C 5%CO2 for 24 hours.
  3. Passage into PMA free medium (6-well plate as an example):
    1. Discard all the medium in the well.
    2. Use 1 mL of PBS to wash the cells 3 times.
    3. Trypsinize HepG2 cells with TrypLE dissociation reagent for 5 min in a 37°C incubator.
    4. Splash the well a few times using TrypLE, check the adherence under the microscope. If the cells are still adherent, incubate for longer.
    5. Add 1 mL of RPMI to stop the trypsinizing.
    6. Prepare a falcon tube and label the cell name on it.
    7. Transfer all 2mL of the cells into the tube.
    8. Centrifugation at 12x100 RPM for 3 min.
    9. Remove as much supernatant as possible without disturbing the pellet.
    10. Add 1 mL/plate (for 6-well plate) of medium to the tube and pipette up and down to resuspend the cells.
    11. Transfer 1000X μL of the cells to the new well.
    12. Add 2000-1000X μL of the RPMI(PMA free) to the well, pipette up and down roughly.
    13. Shake the well in forward-backward and right-left directions 20 times per direction to avoid the clumping.
    14. Incubate the 6-well plate at 37°C 5%CO2.
    15. Check the confluence everyday and passage if necessary.

Microfluidics Lab


Protein Nanoparticles (Zein Nanoparticles)


Preparation of 10mg/mL Zein Solution

  1. Prepare 10 mL of 70% ethanol by mixing 3 mL DI water with 7 mL of 100% ethanol.
  2. Add 100 mg of Zein powder to the 10 mL of 70% ethanol.
  3. Vortex the mixture until fully dissolved.

Production of Zein Nanoparticles via Anti-Solvent Method

  1. Using the anti-solvent method, add 100 µL of the prepared Zein solution into 1 mL of water while vortexing the water continuously.
  2. Continue vortexing for 1 minute to ensure nanoparticle formation.
  3. Centrifuge the mixture to remove larger microparticles and collect the nanoparticles from the supernatant.

Note: Nanoparticles should be used immediately as they will aggregate if left overnight.


Testing Zein Nanoparticles in Different pH Solutions

  1. Prepare 20 mL Tris-HCl solution in a beaker and place it on a magnetic stirrer.
  2. Monitor the pH using a pH meter.
  3. Adjust the pH of the solution by adding HCl to achieve pH levels of 8, 7, 6, 5, and 4.
  4. Collect 6 mL of each pH-adjusted solution for further testing.
  5. For pH levels 9 and 10, adjust the pH of pH 8 Tris-HCl solution by NaOH.
  6. Prepare Zein Nanoparticle by adding 100 µL of the prepared Zein solution into 1 mL of differe

Testing for Different Tween 20 Concentrations

  1. Prepare Tween 20 solutions of 0 mg/mL, 0.5 mg/mL, 1 mg/mL, 1.5 mg/mL, 2 mg/mL, 2.5 mg/mL, and 3 mg/mL by diluting a 5 mg/mL Tween 20 stock solution with DI water.
  2. Vortex the 0.5 mg/mL Tween 20 solution.
  3. Slowly pipette 100 µL of the 10 mg/mL Zein solution into the 0.5 mg/mL Tween 20 solution while vortexing to produce Zein nanoparticles.
  4. Dilute the nanoparticle suspension 10-fold.
  5. Pipette 3 mL of the diluted suspension into a cuvette.
  6. Measure the particle size and zeta potential using the DLS machine.

Testing for Different Centrifugation Time

  1. Prepare Zein nanoparticles under optimal Tween 20 solution (2mg/mL), pH (pH 6).
  2. Add 1.5 mL of Zein Nanoparticles made into three 2mL centrifuge tubes, label the three tubes as tube 1, tube 2, tube 3.
  3. Centrifuge the tubes under 5000 rpm.
  4. At specified intervals during centrifugation (30 seconds, 1 minute, 2 minutes, 4 minutes, 8 minutes and 10 minutes), perform the following steps:
    1. At each time point, stop the centrifuge and remove 150µL of the solution from each tube into a new centrifuge tube.
    2. Immediately return the original tubes to the centrifuge and resume centrifugation.
    3. Repeat this process at each subsequent time point (30 seconds, 1 minute, 2 minutes, 4 minutes, 8 minutes and 10 minutes), transferring 150µL from each tube to new centrifuge tubes at each interval.
    4. By the end of 10 minutes, you will have samples collected at all five time points for further analysis.
  5. Measure the Zein Nanoparticles in terms of Zeta potential, Size, PDI.
  6. Use an Optical microscope to observe morphology.

Encapsulation of Pre-formulated RNA into Zein Nanoparticles for calibration

  1. Prepare 12.86 mg/mL Zein solution (in 90% ethanol).
  2. Prepare 10 mg/mL RNA water solution, pre-treated it by centrifugation with 14800 RPM for 10 minutes to remove insoluble impurities.
  3. Mix 7mL zein solution in 90% ethanol (12.86mg/mL) with 2 mL RNA solution, creating a zein-RNA mixture containing 10 mg/mL zein and 2.22 mg/mL RNA in 70% ethanol.
  4. Prepare RNA-encapsulated zein nanoparticles using the anti-solvent method.

Encapsulation of T7-VEE-GFP RNA from IVT into Zein Nanoparticles

  1. Dissolve and dilute the desired fluorescence-stained srRNA in an appropriate buffer, to prepare a high-concentration srRNA solution (10 mg/mL).
  2. Add Rhodamine B solution to stain the srRNA, producing Rhodamine B (RB)-stained, fluorescence-labeled srRNA in 1000 (RNA) :1 (Rb) ratio.
  3. Dilute the fluorescence-stained srRNA to the following concentrations: 1 mg/mL, 0.1 mg/mL, 0.01 mg/mL, and 0.001 mg/mL, and 0.0001 mg/mL. Use a microplate reader to measure the fluorescence of each concentration and generate a calibration curve. This curve will be used later to quantify encapsulated RNA.
  4. Mix the RB-stained srRNA solution with the optimized Zein NP suspension.
  5. Centrifuge the nanoparticle mixture to separate srRNA-loaded Zein NPs from unencapsulated srRNA for optimized centrifugation time. Collect the supernatant containing the srRNA-loaded nanoparticles.
  6. Quantify the srRNA-loaded nanoparticles in terms of Zeta Potential, Size and PDI.
  7. Measure the fluorescence of the encapsulated RNA using a microplate reader and compare it to the calibration curve to determine the amount of encapsulated srRNA.
  8. Determine Loading Capacity and Encapsulation Efficiency.

Encapsulation of CAR_Ma into Zein Nanoparticles

  1. Prepare optimal Tween 80 solution (2mg/mL Tween 80 and pH 6).
  2. Mix Zein solution (90% alcohol) and the 10µg/µL RNA solution in a 7:2 ratio in volume. In this time, the resulting volume is 400µL.
  3. Anti-solvent method: dropping 400μL zein solution to 4 mL RNA solution (maintaining the 1:10 ratio) while vortexing. Afterwards, vortex for 1 extra minute.
  4. Centrifugation by 5000 RPM for 1 minute (the optimal duration).
  5. Extract the supernatant and discard the sediment.
  6. Quantify the srRNA-loaded nanoparticles in terms of Zeta Potential, Size and PDI.
  7. Measure the fluorescence of the encapsulated RNA using a microplate reader and compare it to the calibration curve to determine the amount of encapsulated srRNA.
  8. Determine Loading Capacity and Encapsulation Efficiency.

Lipid Nanoparticles (NLC)


Preparation of Organic and Aqueous Phases

  1. Organic Phase: In an Eppendorf tube, mix the following components:
    1. 20 µL Squalene
    2. 10 µL DOTAP
    3. 8 µL Span 60
    4. 8 µL DMG-PEG
    5. 2 µL Tri
    6. 52 µL ethanol
  2. Vortex the organic phase mixture and centrifuge briefly if necessary.
  3. Aqueous Phase: In a separate Eppendorf tube, prepare 300 µL of citrate buffer and add 1 µg of your gene therapeutic sample.

Formation of Nanostructured Lipid Carriers (NLCs)

  1. Slowly add the entire organic phase to the wall of the tube containing the aqueous phase while vortexing.
  2. Continue vortexing the aqueous phase to ensure proper mixing and nanoparticle formation.

Note: Ethanol is toxic, and citrate is acidic (pH 4), so avoid using these solutions directly with cells.


Preparation for Cell Experiments

  1. Dissolve the nanoparticles in 150 µL of PBS.
  2. Mix 200 µL of the PBS solution with 5 µL of the nanoparticles to prepare the suspension.

Nanoparticle Characterization using DLS

  1. Add the prepared nanoparticle suspension into a cuvette, ensuring the cuvette triangle is facing you.
  2. Place the cuvette into the DLS machine and select the "IGEM for undergraduates" folder to save the data.
  3. Choose default measurement settings:
    1. Temperature: 25°C
    2. Calibration time: 0 s
    3. Cuvette type: ZEN0040
  4. Measure the sample 3 times, each for 10 seconds.
  5. Record the following data:
    1. Particle size: Optimal range 20-200 nm.
    2. Polydispersity Index (PDI): Optimal range below 0.3.
    3. Zeta Potential: Generally positive for RNA interaction.

References

Chester, N., & Marshak, D. R. (1993). Dimethyl sulfoxide-mediated primer Tm reduction: a method for analyzing the role of renaturation temperature in the polymerase chain reaction. Analytical biochemistry, 209(2), 284-290. https://doi.org/10.1006/abio.1993.1121