TOP10 bacteria supposed to be ccdB sensitive. I transfected them with a gateway plasmid and the selections does not work for me.
can anyone give me some hints? Thanks.
Hi Domokos,
Thanks for sharing this experience. I guess this strain somehow still has resistance to ccdB. You could try One shot OmniMAX2T1 Phage-Resistant competent cells. This strain dhas increased ccdB sensitivity and is recommend for negative selection.
Best,
Wen An
Hello Domokos,
Did you culture your transformed TOP10 in antibiotic selective agar? In that way, you will know that whether you have successfully transformed your bacteria or not. Usually I will do it in that way to make sure that my transformation is successful and the ccdB gene has been replaced with my gene of interest.
Besides, size for gateway plasmid is usually large so recommended to transform using electroporation rather than heat-shock. Hope this can help you.
It could also be that your plasmid has lost the ccdB gene or picked up a mutation. Do you get lots of colonies if you just do an uncut vector only transformation control/?
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