Previously I have cloned estA gene in pET28b with restriction sites EcoRI and BamHI and stored it in -80˚C. I also proceeded with site directed mutagenesis (SDM) after confirmation of the cloning via PCR gene amplification. However, I didn't get positive result post SDM. Now, as I'm trying to repeat the experiment, I'm unable to exact the plasmid from the previously cloned bacteria and stored as glycerol stock. The bacteria shows antibiotic resistance but when I try to extract plasmid and confirm through gel electrophoresis, I'm not getting any band. Please can anyone guide me and help me troubleshoot the problem.
Hello,
After recovering your clones from -80C, have you verified the presence of your cloned gene by PCR of the positive colonies?
Sofiane Benyamina, yes to verify that i wanted to isolate the plasmid, followed by PCR amplification of the gene to confirm the cloned gene. But I,m stuck in the plasmid isolation only. no band is observed after I isolate the plasmid and run on agarose gel.
I asked you the question because I was thinking that maybe antibiotics don't work, but apparently you verified the presence of the cloned gene by PCR.
In this case, perhaps there was homologous recombination and the plasmid was integrated into the genome of the bacteria, this is why you have resistance to antibiotics, the presence of the cloned gene but not the plasmid under extrachromosomal form.
Robert Adolf Brinzer I used Promega's PureYield Plasmid miniprep system kit for isolation of plasmid DNA. other samples are working fine only this particular cloned gene is showing problem
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