The chemocompetent cell strain is derived from Escherichia coli strain B and has the advantage of lacking both lon and ompT proteases. It is currently one of the widely used host bacteria for expression, and its operation method is as follows:
1. Take out the chemically sensitive cells from -80 ℃, quickly insert them into ice, wait for the bacterial block to melt for 6 minutes, add the target DNA (plasmid or ligation product), and gently mix by sliding the bottom of the EP tube with your hand (avoid using a gun to suck). Let it stand in ice for 25 minutes.
2. Heat shock in a 42 ℃ water bath for 45 seconds, quickly place it back on ice and let it stand for 5 minutes. Shaking will reduce the conversion efficiency.
3. Add 0.9 mL of room temperature S.O.C. (S.O.C. is nutrient rich and can improve conversion efficiency) or LB medium to the centrifuge tube.
4. Resuscitate at 30 ℃ and 250rpm for 60 minutes. When the plasmid contains unstable fragments, culturing at 30 ℃ can reduce the probability of erroneous recombination. If the transformation efficiency is calculated by transforming ControlpUC19, it needs to be revived at 37 ℃ and 225rpm for 60 minutes
5. Take out the screening plate in advance and incubate at 30 ℃ to maintain the plate temperature at 30 ℃. Take 50-100 μ l and directly apply it to the screening plate or centrifuge at 5000rpm for one minute to collect the bacteria. Leave about 100 μ l of supernatant and gently blow and resuspend the bacterial block onto LB medium containing the corresponding antibiotic.
6. Invert the plate and incubate at 30 ℃ for 20-24 hours or overnight at 37 ℃. When the plasmid contains unstable fragments, culturing at 30 ℃ can reduce the probability of erroneous recombination. If the transformation efficiency is calculated by transforming ControlpUC19, overnight cultivation at 37 ℃ is required.
Precautions for chemosensory cells:
1. For the cloning of unstable DNA fragments or the construction of retroviral/lentiviral vectors, the coated plates should be cultured at 30 ℃ to reduce the probability of erroneous recombination.
2. When preparing high-purity viral plasmids, fresh transformed agar plates should be used for inoculation, and plasmids should be extracted from fresh bacterial solution. The bacterial solution should not be stored at low temperatures before use.
3. For unstable clones or viral plasmids, priority should be given to storing them in plasmid state, and efforts should be made to avoid storing plasmids in E. coli cells.
4. Chemically sensitive cells generally melt slowly in ice. Insert the target DNA into the ice within 10 minutes, and do not leave it in the ice for too long as it will reduce the conversion efficiency. When mixing with the target DNA, gentle handling should be performed.
5. The conversion of high concentration plasmids or efficient ligation products can correspondingly reduce the amount of bacteria ultimately used for plate coating.