I chemically transformed a ligation product (pET28a and my gene of 3051 bp) into E coli DH5alpha competent cells. I plated 200 and 800 ml of my transformation on plates of LB agar Kan (25 mg/ml). After about 16 h of incubation at 37 °C I get small colonies . Then I transferred the colonies in liquid LB Kan and the growth, carried out at 37°C for 16, 20 h gave a low biomass yield . After miniprep the maximum amount of recombinant plasmid obtained was 13 ng/l. Can you help me to understand why I have a so limited growth of E.coli recombinant cells either on solid and in liquid medium and consequently I obtained a low yield of the pET recombinant plasmid?
Firstly, have you confirmed your cloning by restriction digestion or PCR? DH5a produces small colonies, so that isn't a matter of concern. You mentioned that you have plated 200 & 800ml of your transformations into LB agar plates. ml?
At last from what volume of culture did you isolate the plasmid?
It seems as if your clone is somewhat toxic. First try a control plasmid (pET28 alone) to be certain the problem is not with the strain or the medium. You might also try transforming your plasmid into a different strain to propagate instead of DH5a. Sometimes just changing the strain can help. You might also try growing at lower temperature. You will have to give longer time to grow but this might reduce the toxicity and give you a good cell mass for making plasmd prep.
You can always start by spinning down more cells. How big was your pellet?
Also, make sure that you are shaking vigorously during incubation. E. coli grows fastest when it's well-oxygenated.
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