pLentiCRISPR v1 in E. coli growth

We have some glycerol stocks of E. coli carrying certain pLentiCRISPR V1 plasmids that we would like to grow up and miniprep.

But the bacteria grow badly when streaked out on plates (LB+amp) and not at all when growing up overnight cultures (LB+amp).

Is this a common problem or just us?

Any tips?


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Navy Centauralmost 4 years ago

In my experience, it depends on the strain which you are using.

If using DH5a, there will be less colonies but they will be larger in size.

If using Stbl2/Stbl3, there will be more colonies but the size is quite small (i.e. 1/3 or even 1/5 of "normal" colony) when culturing at 37 degree C for 16h. The size will grow larger when incubating for 24h+

For liquid LB culture, use a lower rpm, ~150 at grow for 20h+.

Double check your colony to see if they really contain the plasmid by colony PCR, also use Amp plates which are not dated.

Good luck.

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