The plasmid containing the pigment protein amliCP was transferred into E. coli DH5α for amplification and incubated overnight.
Take 100μL of freshly prepared receptor cells, add 1μL of recombinant DNA ligation product, mix by blowing on ice, and place on ice for 30min.
Put the tube to 42°C for 90s of heat excitation.
Ice bath for 2min.
Add 800μL of LB liquid medium, incubate at 37°C, 150rpm for lh (slow shaking).
The cells were collected by centrifugation at 12000 rpm for 5min, and about 100 μL of medium was retained. The excess supernatant was discarded, and the cells were resuspended by aspiration and coated in a Petri dish containing chloramphenicol (100μg/mL).
Take 20, 30, 50μl of bacterial solution to be coated in solid medium, respectively, and invert the flat dish at 18:00 and incubate at 37℃, but no colony generation was observed, what could be the reason?
First, the concentration of chloramphenicol (100μg/mL) used in NWU-CHINA-A is high, the concentration of antibiotic we generally use in E. coli is (18μg/mL). Secondly, when you construct the plasmid, the promoter region in front of the chloramphenicol resistance gene was should be checked again.