4 replies to this topic

[Linda Ferreira]

Hi there

I am a Biotechnology student busy with a techniques course. We are doing cloning. Everything has been going well, but for some reason my cells transformed with my recombinant plasmid DNA, as well as those transformed with the ligation control, refuse to grow.

I know my cells are competent as my positive control and all the subsequent dilutions grew just fine. The plates were also made from the same batch of LB broth, amp, tet, IPTG and X-gal. I also have recombinants - did an agarose gel analysis (cloned 1200bp fragment into linearised pBS, EcoRI used as restriction enzyme)

I really don't know what is going on. Did the same procedure twice now, with no growth, blue or white, on those two plates.
Any suggestions will be sincerely appreciated

Regards
Linda

[phage434]

Is your positive control a transformation of plasmid, or is it cells already containing the plasmid?

[Linda Ferreira]

It is a transformation of the pBS plasmid isolated through large scale purification.

[phage434]

OK, you should be able to calculate the transformation efficiency of your cells by counting colonies on dilutions of your plasmid.  You should get 10^7 to 10^8 colonies per microgram of plasmid DNA if you have good efficiency.  Less than that makes it difficult to ligate and then transform.  Many other things can go wrong, but usually it is either poor transformation efficiency or poorly prepared DNA going into the ligation reaction.

[Linda Ferreira]

Thanks for the advice. Calculated the transformation eff and it was quite low.
So, I managed to increase the efficiency and I got a few colonies! So yes, thanks a lot!