         Plasmid extraction

Procedures

1.  Place the absorption column into the collection tube, add 500 μL of buffer S, and then centrifuge with 12000 spins per minute for 1 minute. Remove the waste liquid from the collection tube and place the absorption column back in it.

2.  Centrifuge 1.5 mL of bacterial solution for 2 minutes with 8000 spin per minute. Collect the bacteria in solid form and remove the medium.

3.  Add 250 μL of buffer SP1 and blow it with the pipette until the bacteria is completely suspended.

4.  Add 250 μL of buffer SP2, immediately mix it gently, and let it sit at room temperature for 2-4 minutes.

5.  Add 350 μL of buffer SP3 and immediately mix it gently.

6.  Centrifuge the solution for 5 minutes at 12000 spins per minute. Place the supernatant in the absorption column and centrifuge at 8000 spins per minute for 30 seconds. Remove the liquid from the collection tube.

7.   Add 500 μL of buffer DW1, centrifuge it for 30 seconds at 9000 spins per minute, and then remove the liquid from the collection tube.

8. Repeat step 7.

9. Centrifuge the empty absorption column for 1 minute with 9000 spins per minute.

10. Place the absorption column in a clean 1.5 mL centrifuge tube, add 50-100 μL Elution Buffer (the more buffer added, the lower the concentration), leave it at room temperature for 1 minute, and then centrifuge it for 1 minute. Preserve the DNA solution in the tube.

         Different buffers

1. Buffer SP1

Resuspension buffer to provide an optimum pH for further lysis.

It contains lysosome, glucose, EDTA, and Tris.HCl.

Lysosome is responsible for breaking the cell wall of the bacterial

Glucose is for keeping the solution isotonic

EDTA inactivates many enzymes that may harm the plasmid

Tris.HCl acts as a buffer that maintains the solution at a pH of 8

2. Buffer SP2

This is the lysis buffer.

Contains NaOH and dodecyl sulfate (SDS)

SDS solubilizes the phospholipid and denature the protein.

NaOH provides a highly alkaline condition that denatures the plasmid.

3. Buffer SP3

potassium acetate solution, which has a pH of 4.8. it brings the pH back to neutral and allows the participation of proteins.

         Polymerase chain reaction

Vector PET-Dual

primer A-1F from 5’ and primer A-1R from 3’ for RH3 (failed to obtain) and PPG.

primer A-2F from 5’ and primer A-2R from 3’ for P4H.

Vector PET

Primer C-1F from 5’ and C-1R from 3’ for PKG, DKG and EPG.

Add 25 μL of Phanta Mix 2 μL of primer F and primer R, and 19 μL of water.

Put the mixture in the PCR machine, and it will use 3 different temperatures to unwind and replicate the DNA segment.

         Agar gel electrophoresis

Make use of the fact that DNA is negatively charged at its phospho-group, which means it will move toward the positive charge at different speeds due to the difference in size.

Preparation of 1% agarose gel: 100 mL of TAE, 1 g of agarose and 5 μL of DNA dye.

While adding the DNA sample, the sample should be first mixed with the loading buffer.

A ladder that measures the length of the target DNA will be used.

         Agar gel electrophoresis of the product from 7.27 (same procedures)

         Gel extraction

To obtain target DNA segments from the alga gel once used in the electrophoresis.

1.          Cut the gel containing the target DNA fragment and weigh it out.

2.          Add Buffer B2 that is 3-6 times heavier than the piece of gel. Water bath it at 50°C for 5-10 minutes to melt the gel.

3.          Place the solution in an absorption column in the collection tube and centrifuge it for 30 seconds at 8000 spins per minute. Remove the waste liquid from the collection tube.

4.          Add 500uL of Wash Solution, centrifuge it for 30 seconds with 9000 spins per minute, and remove the waste liquid in the collection tube.

5.          Repeat step 4.

6.          Centrifuge the absorption column for 1 minute with 9000 spins per minute.

7.          Place the absorption column in a clean 1.5mL centrifuge tube and add 15-40uL of elution buffer at the center of the column (the more elution buffer added, the lower the concentration). Let it sit at room temperature for 1 minute, and then centrifuge it for 1 minute. Preserve the DNA solution in the tube.

         Homologous recombination

Homologous recombination of PET24a is for putting the target DNA segment into the vector by enzyme.

Use 2*Clon express reaction system (10uL)

Water bath it at 50°C

         Transformation

To put the plasmid that contains the target gene fragment into a cell/bacteria in order to culture more

1.          Add 10mL of homologous recombination product into the DH5alpha cell

2.          Put it in the ice for 30 minutes

3.          Put the mixture into the water bath for 45 seconds at 42°C

4.          Put it back to the ice for 3 minutes

5.          Add 900mL of LB medium and put it into the shake incubator for 30 minutes at 37°C

6.          Centrifuge the tubes for 10 minutes with 8000 spins per minute

7.          Remove the supernatant, leave 100 uL, and mix them by blowing them with the pipette

8.          Add into solid LB medium in a petri dish

9.          Apply them evenly with a coating rod and cultivate it overnight

The following letters represent one specific plasmid or E. coli transformed using the plasmid.

A: pET-Dual-HisRh3CP15VP4-P4H

B: pET-Dual-HisPPGP15VP4-P4H

C: pET-24a-DKG50-P15VP4

D: pET-24a-PKG50-P4VP4

E: pET-24a-EPG50-P8VP4

A B was successfully cultured; therefore, it will be PCR and be examined by electrophoresis.

C D E wasn’t successfully cultured; therefore, have to be repeated.

Extraction of the plasmid of pET Dual RH3C-P4H failed the first time.

         Solid LB medium preparation

*All amounts of components shown in the table above is the amount required for preparing 1L of both LB mediums.

Step 1: weigh out each component.

Step 2: mix the components together and dissolve them in distilled water.

Step 3: seal the container of the solution with aluminum foil

Step 4: autoclave it for 2 hours with a maximum temperature of 120°C

         Plasmid extraction

1. Place the absorption column into the collection tube, add 500 μL of buffer S and then centrifuge with 12000 spins per minute for 1 minute. Remove the waste liquid in the collection tube and place the absorption column back in the collection tube.
2. Centrifuge 1.5 mL of bacterial solution for 2 minutes with 8000 spin per minute. Collect the bacteria in solid form and remove the medium.
3. Add 250 μL of buffer SP1, blow it with the pipette until the bacteria is completely suspended.
4. Add 250 μL of buffer SP2, immediately mix it genteelly and place it in room temperature for 2-4 minutes.
5. Add 350 μL of buffer SP3, immediately mix it genteelly.
6. Centrifuge the solution for 5 minutes with 12000 spin per minute. Place the supernatant in the absorption column and centrifuge with 8000 spin per minute for 30 seconds. Remove the liquid in the collection tube.
7. Add 500 μL of buffer DW1, centrifuge it for 30 second with 9000 spin per minute and then remove the liquid in the collection tube.
8. Centrifuge the empty absorption column for 1 minute with 9000 spin per minute.
9. Repeat step 8
10. Place the absorption column in a clean 1.5 mL centrifuge tube, add 50-100 μL Elution Buffer (the more buffer added, the lower the concentration), place it in room temperature of 1 minute and then centrifuge it for 1 minute. Preserve the DNA solution in the tube,

         Culture Bacteria (C D E)

1. Add 5 μL of the cells into the petri dish that is covered by solid LB medium.
2. Add 10 μL of liquid LB medium at the point where the cell is added
3. Spread them evenly on the plate with the coating rod.
4. Culture them overnight

         PCR

PET dual

1. MCS1 multiple cloning site 1 RH3CVP4

2. MCS2 multiple cloning site 2 P4H~750bp

A. Make a new medium and draw a grid.

Only take a separate colony.

Restripe the bacteria that were cultivated yesterday into the newly prepared medium.​

Repeat this step four times.

keep the environment 37℃.

B. colony verification (PCR + agarose gel electrophoresis, AGE）

The reaction system has a total of 100 μL

5’ Primer→4 μL

3’Primer→4 μL

2x PCR Mix →50 μL

add water→to 50 μL

Pour 100 μL of colonies into the test tube on average 5 times.

3. PCR (2x PCR Mix)

Denaturation 94℃ 5 min

Degeneration 94℃ 30 s

Annealing 50-60℃ 30 s

Stretch 72℃ 30-60 sec/kb

Final Stretch 72℃ 10 min

Degeneration → Stretch（30-35 cycle）

4. Make agarose gel, wait for 20 minutes until it solidifies, and put the PCR product into

the well. Wait 30 minutes.

         Electrophoresis of A B that are previously PCR

Agarose gel preparation

1. Mix 100mL of TAE with 1g of agarose gel.
2. Seal the container with aluminium foil.
3. Heat it in a microwave for 3 minutes until it resolves.
4. Add 5uL of DNA dye and mix.
5. Pour the solution into the model and wait until it cools down and forms solid.

While adding the DNA sample, the sample should be first mixed with the DNA loading buffer.

A ladder that measures the length of DNA in BP will be used.

Electrophoresis for 30 minutes with 120 V.

         PCR of B\C\D\E that was cultured (PPG DKG PKG EPG)

Denaturation 94℃ 5min

Degeneration 94℃ 30s

Annealing 50-60℃ 30s

Stretch 72℃ 30-60sec/kb

Final Stretch 72℃ 10min

Degeneration → Stretch （30-35 cycles）

100 μL PCR reaction system

         Culturing bacteria

pick the bacteria from the petri dish

put them into the tube that contains LB medium

culture it overnight

         Electrophoresis of plasmid B (HisPPGP15VP4-P4H) and plasmid E (EPG50-P8VP4)

         Culture plasmid A-E in BL21

         SDS-Page electrophoresis gel preparation

         PCR verification of the A-E in BL21

         0.2 mM IPTG (Isopropyl-beta-D-thiogalactopyranoside) inducing protein expression in BL 21.

         Electrophoresis

Gel preparation

1. Mix 100 TAE with 1g of agarose.
2. Seal the container with aluminium foil.
3. Heat it in the microwave for 3 minutes until it boils.
4. Add 5uL of nuclei acid dye and mix.
5. Pour the solution into the electrophoresis container and wait until it cools down and forms a solid.
6. Load the 2K ladder so the DNA can be measured in BPs.
7. Mix the DNA sample with the loader and inject it into the gel.
8. Electrophoresis it for 30 minutes with 120 V.

         Culture plasmid A-E in BL21

Select the bacteria colony from the petri dish, Add it to the tube containing LB medium, Shake it for 7h

         SDS-Page electrophoresis gel preparation

1.      Prepare the lower-layer gel by adding the same amount of lower-layer gel solution and buffer.

2.      Inject them into the container carefully to form a flat surface.

3.      Add double distilled water to the surface to keep it flat.

4.      When the lower layer of gel is solidified, pour away the water.

5.      Mix the lower layer gel buffer and solution.

6.      Inject it carefully into the container.

7.      Push the comb inside the upper layer.

8.      Leave it at room temperature for 20 minutes until it solidifies.

9.      Keep it moist and store in 4°C.

         PCR verification of the A-E in BL21

Denaturation 94℃ 5min

Degeneration 94℃ 30s

Annealing 50-60℃ 30s

Stretch 72℃ 30-60sec/kb

Final Stretch 72℃ 10min

Degeneration → Stretch （30-35 cycles）

100uL PCR reaction system

primer A-1F from 5’ and primer A-1R from 3’ for RH3

primer A-2F from 5’ and primer A-2R from 3’ for P4H.

Add this reaction system to the plasmid extraction.

         IPTG promote protein expression

IPTG diluted to 0.2 mM

         Protein purification

         Electrophoresis of the purified protein using the SDS-PAGE gel prepared on 7.31.

         Purification of the protein

1. Collect the bacteria solution promoted overnight.
2. Centrifuge it, remove the supernatant (LB medium) and weigh the remaining bacteria.
3. Add lysis buffer; 1 g of bacteria should be mixed with 4 mL of lysis buffer.
4. Mix it evenly and use ultrasonication to break down the bacterial wall.
5. Centrifuge the solution and collect the supernatant, place it on the ice surface.
6. Add the lysis buffer and His-tag purification resin into the empty affinity chromatography column.
7. Add the supernatant in the column, collect the run-through fluid and repeat 3-5 times.
8. Wash the column with a wash buffer 5 times.
9. Elute the bound proteins with a wash buffer containing imidazole 3 times.

         Electrophoresis of the purified protein

1.      Inject the sample of protein into the gel

2.      Electrophoresis it with 100 V till it passes the upper layer, then use 200 V.

3.      Take the gel and submerge it in the Coomassie Blue Staining solution.

4.      Wash it in the destaining solution overnight.

         Use native PAGE electrophoresis (10% of separation gel) to examine the protein.

Total 15mL of separation gel.

1.      Add 6.1mL of distilled water.

2.      Add 5 mL of 30% Arc-Bis.

3.      Add 3.75 mL of gel buffer A.

4.      Add 10 % APS for 0.15 mL.

5.      Add 0.006 mL of TEMED and pour the mixture into the gel container.

6.      Wait for the separation gel in the container to solidify.

7.      Prepare the concentration gel.

8.      Add 1.33 mL of distilled water.

9.      Add 0.67 mL of 30% Arc-Bis.

10.  Add 2 mL of gel buffer B.

11.  Add 0.04 mL of 10% APS.

12.  Add 0.004 mL of TEMED.

13.  Pour it in the container after the separation gel is solidified and then push the comb in the gel.

14.  After the concentration gel solidifies, remove the comb and load the sample.

15.  Electrophoresis for 20 minutes with 80 V and then use 100 V till the end.