Im making competent cells from Shigella with pkD46 plasmid already inside it. Im then electroporating them to get donor DNA go inside them. The efficiency of recombination is pretty low for Shigella. Is there any method in which i can increase the competency of my competent cells ?
Are you trying to transform another plasmid or DNA to achieve recombination? If you are trying recombination then..... Problem might be other than cell. As recombination is very difficult to achieve and also getting correct insert is difficult. Can you please elaborate?
Im trying to achieve recombination with a donor DNA with the help of Lamda red genes for aiding homologous recombination. I have tried it a couple of times but Im not getting any mutant colonies. Can you suggest anything? Rajindra Napit
Having lamda DNA helps recombination by decreasing DNA degradation due to cellular DNAse, however it is not a compulsory to have it for a successful recombination. Recombination can sometimes get very difficult.... So i would suggest you to make sure you have designed primer/DNA with sufficient homologus regions (at least 50bp). Having large quantity of DNA(I noticed PCR products have higher success rate than donor DNA due to more DNA) for transformation increases the chance of success. In this type of experiment doing more no. Of transformation is a good strategy to get result quickly. hope my answer helps!
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