I am facing a problem with the plasmid from addgene (pcW57.1), when transformed into chemically competent DHFa cells, did not give any single colony overnight in ampicilin containing agar media.
https://www.addgene.org/browse/sequence/181675/
I am planning to remove ccdB gene by Nhe1 and Sal1 cuts and then by blunt ended ligations I will transform again. Will this be correct approach? (Later I will insert our GOI with blunt ends too)..
Kindly anyone with experience of working with this plasmid and or system, please share your opinion.
Hi Aadil,
I think the approach you mentioned would not create any blunt ends, as both Nhel and Sal1 will make sticky ends. The best approach for this would be to buy CCDB tolerant Bacterial strain, or to use a PCR mediated deletion of CCDB cassette if you cannot buy the cells.
I hope you would have solved this problem by now, but just in case if you require any suggestions please let me know.
Regards
KJ.
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