Difference between revisions of "Team:UCAS-China/People/Notebook"

Notebook can be downloaded here

Preparation of competent cells

1. Streak competent cells on LB plates, and incubate overnight.

2. Pick a single colony and incubate at 37°C in 4ml LB medium.

3. Add 100l of bacteria solution to 100ml LB medium in conical flask.

4. Shake the flask at 37 °C until the OD600 reaches 0.4-0.5, about 3h.

5. Place the conical flask quickly on ice, violently oscillate it to cool it down quickly.

6. Transfer the medium to a pre-cooled centrifuge tube, centrifuge at 4500 rpm for 10 min, and discard the supernatant.

7. Add 2/3 volume of pre-cooled CaCl2-MgCl2 mixture to each tube, resuspend the cells, and ice bath for 10 min.

8. 4 degrees, centrifuge at 4500rpm for 10min, completely discard the supernatant

9. Add 1/25 volume of pre-cooled 0.1M CaCl2 solution to each tube, resuspend the cells, and ice bath for 10 min.

10. Add 7% volume of pre-cooled DMSO to each tube, ice bath for 10 min, dispense into 1.5 ml EP tube, dispense 100 per EP tube, and store in -80 refrigerator.

Bacteria strain preservation

1. Mix 900 bacteria solution with 900 glycerin (60%)

2. Store the mixture in a -80 degree refrigerator

Chemical transformation

Material:

Plasmid solution to be transformed

Competent DH5α cells

LB broth

Selection plates

Ice

1.5mL Microtubes

Methods:

1.Pipette 50µl of competent cells into pre-chilled 1.5ml tube: 50µl in a 1.5ml tube per transformation. Tubes should be labeled and pre-chilled. Keep all tubes on ice.

2.Pipette 4µl of resuspended DNA into 1.5ml tube: Gently pipette up and down a few times. Keep all tubes on ice.

3.Close 1.5ml tubes, incubate on ice for 30min.

4.Heat shock tubes at 42°C for 90 sec: 1.5ml tubes should be in a floating foam tube rack. Place in water bath to ensure the bottoms of the tubes are submerged. Timing is critical.

5.Incubate on ice for 3 min: Return transformation tubes to ice bucket.

6.Pipette 700µl LB media to each transformation.

7.Incubate at 37°C for 1 hours, shaking at 220 rpm

8.Spin down cells at 4000 rpm for 2mins and discard 550µL of the supernatant. Resuspend the cells in the remaining 200µL, and pipette each transformation onto selection plates with appropriate antibiotics. Spread with sterilized spreader or glass beads immediately.

9.Incubate transformations overnight (14-18hr) at 37°C: Incubate the plates upside down (agar side up).

10.Pick single colonies: Pick single colonies from transformations: do a colony PCR to determine whether the transformation is successful.

Colony PCR

Material:

Sterile Water

Selective LB broth

12.5 µL 2*SuperfastTaq mastermix

1 E. coli colony

1 µL of 10 µM forward primer

1 µL of 10 µM reverse primer

Methods:

1.Pick single colonies and incubate 150 µL LB broth in a 96-well plate.

2.Incubate 2 hours at 37°C, 1000rpm.

3.Combine 0.5 µL cell culture, 12.5 µL 2*SuperfastTaq mastermix, 1 µL of 10 µM forward primer, 1 µL of 10 µM reverse primer, and sterile water up to 25 µL.

4.Incubate in the thermocycler, the settings are as follows.

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PCR from template

Material:

5x FastPfu buffer

10 µM forward primer

10 µM reverse primer

PCR tube

Sterile water

Template DNA

Methods:

1. In a PCR tube, combine 1 µL of plasmid DNA, 2 µL of 10 µM forward primer, 2 µL of 10 µM reverse primer, 10 µL of 5x FastPfu buffer, 1.5 µL of 10 mM dNTP mix, 0.5 µL of FastPfu and sterile water up to 50 µL.

2. Gently mix the reaction

3. If necessary, collect the liquid to the bottom of the PCR tube by spinning briefly

4. Transfer the PCR tube from ice to a PCR machine preheated to 98°C to begin thermocycling The settings are as follows.

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DNA gel electrophoresis

Material:

Agarose Powder

TAE buffer

Gel mould

GoldView

Gel Tank

DNA ladder

DNA loading dye

Methods:

1.Prepare 1% w/v solution of agarose powder in 1x TAE buffer using a conical flask

2.Heat the mixture until agarose is completely dissolved.

3. Add 0.01% GoldView to the solution. Make sure there are no bubbles in the solution.

4. Pour the solution into a gel mould

5.Allows the solution to set (approx 15-20 minutes)

6.Transfer the agarose gel to a tank, remove the comb and apply:

4 µL of the DNA ladder 4 µL of DNA samples with the corresponding amount of DNA loading dye (6X)