1Thaw 50 µL competent E. coli cells on ice for 10 minutes
2Add 5 µl DNA from a ligation reaction mix
3Carefully flick the tube 4-5 times to mix cells and DNA.
4Place the mixture on ice for 30 minutes.
5Heat shock at exactly 42°C for exactly 30 seconds.
6Immediately place the mixture on ice for 2 minutes.
7Pipette 950 µL of LB media into the mixture.
8Incubate in the 37°C shaker for 60 minutes.
1Pellet cells at 6000 rpm for 2 minutes;
2Remove and dispense 750 µL of supernatant;
3Re-suspend cells by light vortexing;
4Plate resuspended cells.
10Incubate overnight at 37°C with plates upside down.
Growing Overnight Cultures
12 ml culture tube
Methods:Overnight cultures were prepared under sterile conditions using a alcohol burner.
1Add 5 ml liquid LB media into 12 ml culture tubes.
2Add 5 μl of appropriate antibiotic into the broth.
3Pick a single colony and inoculate the cultures into the LB broth.
4Seal the tubes and incubate in the 37°C shaker.
1 E. coli colony
10 µM forward primer
10 µM reverse primer
1Combine 1.6 µL of dNTP (25mM), 1 µL of DMSO, 0.2 µL of rTaq enzyme, 2 µL of 10X buffer, 0.5 µL of 10 µM forward primer, 0.5 µL of 10 µM reverse primer, and sterile water up to 20 µL.
2Dip a sterile toothpick to dip in a single colony, and dip it in the mixture to add the cells. Note: Do not add too much cells.
3Incubate in the thermocycler.
ThermocyclingThe PCR machine should be set to run the following steps:
|Initial denaturation||95||10 min|
|Final extension||72||10 min|
Preparation of LB Broth and Agar
1Add 10 g Tryptone, 5 g yeast extract and 10 g NaCl to 1 litre purified water.
2Stir to thoroughly mix.
1Add 15g agar, 10 g Tryptone, 5 g yeast extract and 10 g NaCl to 1 litre purified water.
2Stir to thoroughly mix.
DNA Gel Electrophoresis
8-10 µL DNA ladder
DNA loading dye
1Prepare 8% w/v solution of agarose powder in 1/10 TAE buffer (e.g. 0.8g agarose powder in 100 mL buffer) using a conical flask.
2Heat the mixture until agarose is completely dissolved. Do not let the solution boil.
3Pour the solution into a gel mould.
4Add 3 µL EB dye to the solution. Make sure there are no bubbles in the solution.
5Allows the solution to set (approx 15-20 minutes).
6Transfer the agarose gel to a tank, remove the comb and apply:
8-10 µL of the DNA ladder
DNA samples with the corresponding amount of DNA loading dye.
7Run the gel for 30-45 minutes at 100V.
1 µL T4 DNA ligase
1 µL 10X T4 DNA ligase buffer
3 µL vector plasmid
5 µL insert DNA (the ratio of plasmid and DNA should be modified according to the concentration of the DNA)
1Add vector plasmid, insert DNA, T4 DNA ligase and T4 DNA ligase buffer to the microcentrifuge tube on ice (add T4 DNA ligase last).
2Make reaction up to 10 µL using sterile water.
3Flick the tube 4-5 times to mix.
4Incubate at 16°C for at least 30 - 60 min for sticky ends or 1-2 hours for blunt ends.
Materials:Restriction Enzyme: Choose the enzymes according to target sites. Fast Digestion BamHI and XhoI are used for assembly of pET28a.
1Combine 1 µL of restriction enzyme 1, 1 µL of restriction enzyme 2, 2 µL of buffer, 10 µL of plasmid DNA, and sterile water up to 20 µL.
Note: the volume of plasmid DNA can be slightly increased if the concentration is too low. The concentration of plasmid DNA in the mixture should be 0.1-0.4 µg/µL.
2Incubate digestion reaction at 37°C water bath for 30 minutes.