I am using NEB PCR cloning Kit. There is pMiniT vector inside kit. I transformed ligation bwt my PCR product and pMiniT vector to NEB 10-beta competent E.coli cells. I used SOC growth medium for transformation to increase efficiency. I got colony on LB-agar plate. I inoculated some of these colonies in LB medium. However, none of my colonies didnt grow. Should I use different growth medium?
either the bacteria need certain antibiotic to grow (depends on the kit), or you simply innoculated a very small amount and it needs more incubation time to appear!
Generally, E. Coli must grow on LB medium. Maybe your LB-agar media have problem, I mean the material you use to make the media, or the amounts you put. Check your material and method.
You can try other media but are you sure your wire loop was not too hot at the time of picking the colonies?
Another possibility is that your plate was bad and colonies did not contain vector. When inoculated in LB broth containing antibiotics, the empty colonies failed to grow.
LB is the most friendly medium for E coli. Even in spent LB medium E coli growth takes place properly.
What is the antibiotic you used for selection. You may want to grow Transformed Ecoli in LB without antibiotic to troubleshoot as mostly a little high concentration of antibiotic can also lead to this effect.
I had similar troubles while growing caulobacter in PYE.
Thank you for your answers
I used ampicilin. These cells are ampicilin resistant. I used 50ul/ml as final concentration. In protocole, 100ug/ml is recommended. However, I dont know increasing ampicilin concentration works. I can try 100ul/ml ampicilin and incubating longer.
For people searching for this in the future. Look to see if you plated to much of the cells.... I think that may be the issue here.
The NEB PCR cloning kit selection method relies on a toxic mini gene that will be reconstituted if the linear pMiniT2.0 plasmid ligates without an insert. The selection against the toxic mini gene carrying recircularized pMiniT2.0 works best when the number of colonies on the plate is
@Ezgi Odabasi... I am also having the same problem in my research now. Please can you kindly share how you overcame this challenge?
@James Mann...Thank you very much for that valuable information.
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