1. AGAROSE GEL ELECTROPHORESIS

INGRIDIENT

a) Agarose

b) 1X TAE buffer

c) Samples

d) Goldview™ 

e) 10X loading buffer

f) DNA marker (50b or 10kb, depending on the samples)

PROCEDURES

1. Obtain an electrophoresis chamber.

2. Place a ten-tooth comb in the middle set of notches of the gel-cast bed. There should be a small space between the bottom of the teeth and the bed.

3. Mix a 1% (weight by volume) mixture of agarose powder in a sufficient volume of buffer to fill the gel chamber. Heat the mixture until the agarose dissolves.

4. When the hot agarose solution has cooled to 50°C, add Goldview into the agarose solution and pour the agarose solution into the gel-cast bed.

5. After the gel has solidified, gently remove the comb by pulling it straight up.

6. Submerge the gel under the buffer in the electrophoresis chamber.

7. Mix the samples with corresponding amount of loading buffer

8. Use a micropipette to load the samples into the wells of the gel.

9. Carefully snap on the cover of the electrophoresis chamber. The red plug in the cover should be placed on the terminal indicated by the red dot. The black plug in the cover should be placed on the terminal indicated by the black dot.

10. Turn on the power and set the voltage at 90 V.

11. After 30 min, turn off the power and disconnect the leads from the power source. Gently remove the cover from the chamber.

12. Use Gel Image Station to analyze the gel, rename the photograph captured with date and sample name.

2. SDS-PAGE and Western Blot

INGRIDIENT

1. ddH₂O

2. Acrylamide

3. Tris-HCL solution（）

4. Isopropanol

5. 10% AP solution

6. 30% SDS solution

7. TEMED solution

8. 1 X Tris-glycine running buffer

9. Loading buffer

10. PVDF membrane

11. Transfer buffer

12. Blocking solution (5% powdered milk in TBS-T)

13. TBS-T

14. Milk powder

15. Primary antibody (Histidine-tag)

16. Secondary antibody (goat anti-mouse antibody)

17. Bio-Rad Clarity ECL reagent

18. Film

PROCEDURES

1. Gel making

a) Assemble the Bio-Rad apparatus, with the short plates toward the center.

b) Prepare the gel using the formula showing below.

c) Pour the stacking gel into the chamber, the volume of stacking gel is about 60% of the volume of chamber.

d) Add Isopropanol to seal the stacking gel, and when stacking gel is solidified after about 10-20minutes, pour out the isopropanol, use filter paper to clear the chamber.

e) Pour separating gel into the chamber, when it solidified, we can prepare to load samples in gel.

2. Electrophoresis

a) Two gels will be run per apparatus

b) Attach the electrodes to the power supply.

CAUTION: Be sure to match the anode (red) with the positive lead and the cathode (black) with the negative lead.

c) Turn on your power supply. Run your gel at constant voltage (120 V). If you have multiple gels attached to your power supply, you do not need to change the voltage.

d) Run the gel until the loading buffer reaches the bottom of the gel.

3. Transferring to PVDF membrane

a) Prepare the transfer buffer as showing below (Per 1 liter), use magnetic stirring to mix the buffer

i. Tris base:3.03g

ii. Glycine:14.4g

iii. ddH₂O:800 ml

iv. methanol:200 ml

b) pretreat the membrane with methanol, soak the membrane into methanol to activate it.

c) Assemble a “gel sandwich” as showing below ⁽²⁾

d) Put the gel holder cassette with assembled sandwich into the buffer tank, remember to put two cassettes in a tank.

e) Place the cassette in the module. The gray side of the cassette should face the black side of the module, and the clear side of the cassette should face toward the red side of the module. 

⁽²⁾

f) Put the module and cooling units into the buffer tank.

g) Fill with transfer buffer to the top

h) Turn on the current to 300 mA for 90 minutes.

4. Blocking

a) When the electrophoresis is finished, take the membrane out of the cassette, prepare some milk-TBST solution as shown.

5% powdered milk in TBS-T

b) Put the membrane in milk for 1 hour on a platform shaker.

5. Incubation with Primary antibody

a) Take the membrane out of the container, and prepare His-Tag, the primary antibody.

b) Make a dilution of 5000:1, which is 5ml of milk-TBST to 1μl of antibody.

c) Add the solution and the membrane into sealed plastic films, and incubate on platform shaker overnight (about 10-12 hours).

6. Incubation with Secondary antibody

a) The next morning, take the membrane out of the plastic films, wash the film with T-BST solution in a container for three times, with a duration of ten minutes each.

b) Make a dilution of 5000:1, which is 5ml of milk-TBST to 1μl of antibody.

c) Add the solution and the membrane into sealed plastic films, and incubate on platform for 1 hour.

7. Enhanced Chemiluminescence

a) Make a mixture of the developing solution.

b) Drop the solution on the membrane, be sure to soak the whole membrane in the solution.

c) If exposing by film, wrap the membrane carefully in plastic wrap before exposing to film in a darkroom.

d) Record the result of the exposed film.

3. PCR

INGRIDIENT

1. Template DNA

2. Forward primer

3. Reverse primer

4. Deoxynucleotide triphosphates (dATP, dCTP, dTTP, dGTP)

5. Thermostable DNA polymerase

6. Buffer

7. Magnesium (included in the buffer)

8. ddH₂O

PROCEDURES

1. Prepare several 2mL Eppendorf tubes.

2. Using pipette tips, pick several single colonies from the agar gel medium into individual tubes. Fully vortex the mixture to suspend the bacterium.

3. Make up a 50μL PCR system in PCR tubes

a) 10× Buffer: 5μl

b) dATP, dCTP, dTTP, dGTP: each 200μM

c) forward primer: 0.25μM

d) reverse primer: 0.25μM

e) template: 100ng

f) polymerase: 1U

g) ddH₂O: bring final volume to 50μl

4. Set the PCR protocol to “Colony PCR”, place the tubes into the PCR machine and lock the heat cover. Start the protocol.

5. Protocol of Colony PCR: 95 ℃  preheating for 2 minute;30 replicates of these steps: 95 ℃  heating for 30 seconds, 55 ℃  annealing for 30 seconds and 60 ℃  extending for 1minutes  72℃； 6. 

4. Plasmid DNA extraction

INGRIDIENT

1. OMEGA E.Z.N.A. Plasmid Mini Kit

2. ddH₂O

PROCEDURES

1. Transfer 5ml of E.coli with plasmid into Luria-Bertani (LB)/kanamycin plate with antibiotics, culturing incubated for 8-12h at 37℃ with vigorous shaking (¬300 rpm).

2. Obtain 1.5-5 mL of the culture in a microcentrifuge tube.

3. Harvest the bacterial cells by centrifugation at 10000 rpm for 3 minutes.

4. Remove all traces of supernatant by decanting or micropipetting the media into a liquid waste container with 10% bleach.

5. Resuspend the bacterial pellet in 250μL buffer P1(Resuspension buffer). The bacteria should be resuspended completely until no cell clumps remain.

6. Add 250 μL buffer P2, mix gently but thoroughly by inverting four to six times. Continue to inverting the tube until the solution becomes viscous and slightly clear.

7. Add 350 μL Buffer N3 to the lysate and mix immediately and thoroughly and gently by inverting four to six times. After addition of buffer N3, a fluffy white precipitate containing genomic DNA, proteins, cell debris, and SDS becomes visible. The buffer must be mixed completely. If the mixture still appears viscous and brownish, more mixing is required to completely naturalize the solution.

8. Centrifuge for 10 minutes at 13000 rpm in a microcentrifuge.

9. Apply the supernatant (liquid above pellet) from step 8 on the QIA-prep spin column (containing the silica membrane) by pipetting, taking care not to transfer the fluffy white precipitate. Place column in accompanying tube.

10. Centrifuge for 30 seconds. Discard flow-through.

11. Wash the QIA-prep spin column by adding 0.5 mL Buffer PB and centrifuge for 30 seconds. Discard the flow-through.

12. Wash the QIA-prep spin column by adding 0.75 mL Buffer PE and centrifuge for 30 seconds.

13. Discard flow-through, and centrifuge for an additional 1 minutes to remove residual wash buffer.

14. Place the column in a clean 1.5 mL microcentrifuge tube. To elute DNA, add 50 μL Buffer EB (Elution Buffer) to the center of each column, let stand for 1 minutes, and centrifuge 1 minutes. (If necessary, cut the lid off the 1.5 mL tube with scissors so that it will fit in the microcentrifuge.)

15. Your pure DNA is at the bottom of the tube! Remove and dispose of the column and label the tube with “uncut pET-41a”, the date, and your initials and transfer the DNA to the labeled tube.

Reference

1. Biology Laboratory Manual.McGraw-Hill.11^th (2017)

2. Molecular Biology Techniques. A Classroom Laboratory Manual.Elsevier.4^th (2019)