Stbl3 transformants did not grow in LB broth, any suggestion?

[Simon]

We cloned gRNAs into lentiCRISPRv2 and transformed Stbl3. when we picked single colonies to inoculate LB broth with 100ug/ml ampicillin, no growth was seen. We tried to decrease amp to 50ug/ml, there was still no growth. Did anyone have this problem? 

[Simon]

Help please. Thanks.

[Nadia Hassounah]

I have not had this problem. Did the positive control grow? Did you grow the bacteria on plates that were also at 100ug/ml amp? 

[Simon]

Nadia, Thanks for your comments. They grow on LB agar with 100ug/ml Amp but when inoculated in LB broth they won't grow.

[Le Cong]

Hi Simon and Nadia, one thing I think you could check is, because pLentiCrispr is large vector, and stbl3 is a slow-growing strain, it might be that the growth of the transformed stbl3 is slow so after overnight (12 hours) incubation the density of the E coli is still low and might appear to be fairly clear. You should try to wait around 18-20 hours and check if the culture becomes turbid. 

Best,

Le

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[Simon]

Le, thanks for your suggestion. We do have tried to culture for over 30 hrs but see no difference. We may have to start over from ligation or transformation. Thanks again. Simon