Protocols and methods

Plasmid Construction

In our experimental design, plasmid construction is the basis of all work.In the construction of plasmid, we mainly did the following work:

Obtain the desired plasmid from the distribution kits:

1. With a pipette tip, punch a hole through the foil cover into the corresponding well of the part that we want.
2. Pipette 10µL of dH2O (distilled water) into the well. Pipette up and down a few times and let sit for 5 minutes to make sure the dried DNA is fully resuspended. The resuspension will be red, as the dried DNA has cresol red dye.
3. Transform 3µL of the resuspended DNA into competent cells, plate the transformation with the appropriate antibiotic* and grow overnight.
4. Pick a single colony and inoculate broth (again, with the correct antibiotic) and grow for 16 hours.
5. Use the resulting culture to miniprep the DNA and make our own glycerol stock.

Using the miniprepped DNA to run QC tests, such as restriction digests and sequencing.

Glycerol stock

1. Add 0.1 ml of 80% glycerol in H 2 O to a cryogenic vial.
2. Add 0.4 ml sample from the culture of bacteria to be stored.
3. Gently vortex the cryogenic vial to ensure the culture and glycerol is well-mixed.
4. Use a tough spot to put the name of the strain or some useful identifier on the top of the vial.
5. On the side of the vial list all relevant information - part, vector, strain, date, researcher, etc.
6. Store in a freezer box in a -80 ℃ freezer.

Make Competent Cells

(Most of the time we use commercialized competencies DH5 alpha)

1. Add 200μL E.coli DH5 alpha to LB medium and row the cells on a shaker at 37 ℃ for 4 hours
2. Take 1.5mL of the culture product and add it to the EP tube. Centrifuge at 4000rpm for five minutes 4 ℃ . Discard the liquid and add 0.6ml of CaCl 2 solution. Ice bath After 30 minutes
3. Centrifuge at 4000rpm for five minutes 4 ℃ . Discard the liquid and add 80μL of CaCl2 solution

Single Tube Transformation

1. Thaw competent cells on ice
2. Pipette 3µl of resuspended DNA into 100µl thawed cells. Gently pipette up and down a few times. Keep all tubes on ice.
3. Incubate on ice for 25min.
4. Heat shock tubes at 42°C in water bath for 45 sec.
5. Incubate on ice for 5min.Return transformation tubes to ice bucket.
6. Pipette 500µl LB media to the transformation.
7. Incubate at 37°C for 1 hours, shaking at 220rpm
8. Spin down cells at 6000rpm for 1mins and discard 400µL of the supernatant. Resuspend the cells in the remaining 100µL, and pipette the transformation onto petri plates Spread with sterilized spreader.
9. Incubate transformations overnight (14-18hr) at 37°C: Incubate the plates upside down (agar side up).

Colony PCR

1. Prepare several 0.2mL Eppendorf tubes with 20μL ddH2O(double-distilled water) in each.
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 10μL PCR system in PCR tubes(5μL PCR Mix Master, 3μL ddH2O, 1μL suspension and 1μL of primer mix)
4. Set the PCR protocol to “Colony PCR” *, place the tubes into the PCR machine and lock the heat cover. Start the protocol.
*Protocol of Colony PCR: 94 ℃ preheating for 5 minute;30 replicates of these steps: 94 ℃ heating for 30 seconds, 55 ℃ annealing for 30 seconds and 72 ℃ extending for 1minutes(at a speed of about 1 kbp/min) ； keep the products at the temperature of 16 ℃ .

Plasmid DNA extraction （lifefeng® ）

1. Transfer 2mL of the culture to a Eppendorf tube. Centrifuge at 13000rpm for 1 minutes. Discard the supernatant. Add 250μL of resuspension solution(P1 buffer). Completely resuspent cell pellet.
2. Add 250μL of lysis solution(Buffer P2) and mix gently. Set aside for 1 minute.
3. Add 350 μL of neutralizing solution(P3 buffer) and mix by inverting the tubes for 5-10 times. Buried in the ice for 10 minutes.
4. Centrifuge at 12000rpm for 10 min and carefully transfer the supernatant to a adsorption column. Centrifuge at 10000rpm for 30sec, and remove the supernatant.Add 500μL of W1 Buffer and centrifuge at 10000rpm for 30sec.
5. Add 500μL of W2 Buffer and centrifuge at 10000rpm for 30sec. Repeat twice.
6. Centrifuge for 1min and open the lid for 1min to make the alcohol evaporate completely. Move the adsorption column to a new 1.5ml Eppendorf tube and add 50 μL of H2O to obtain the plasmid DNA.

Gel extraction

1. Cut the DNA stripes from the gel under the Blue LED light （ keep the gel thin when you make it. ）
2. Use lifefeng® Gel Extraction Kit to extract target sequence from the gel just cut off.

Utilize PCR to acquire target sequences

PCR system:

2*Master Mix 5μL
ddH2O 3μL
Template 1μL
Forward primer 0.5μL
Reverse primer 0.5μL

PCR procedure:

94 ℃  5min
30 cycles (94 ℃  30s,55℃  30s,72 ℃  1kb/min ）
72 ℃  5min

Team:ASTWS-China/Protocols