One of my colleagues working on microbial isolation facing the problem regarding the 16S amplification for microbial cultures. Same samples were amplified for Phylotyping but unable to amplify via 16S primer please anyone suggest what I have to do? If possible, please also share the optimized conditions for Pathotyping of E. coli.
Can you tell us more about your PCR conditions ? Normally, amplifying 16S on E. coli does not pose any particular problem. What primers do you use, what is the nature of the template ?
E.coli was morphological identified on the selective media (McConkey, EMB media), PCR conditions for 16S E.coli primers are 95C for 3mins, 94C for 45secs, 58C for 45 secs, 72C for 1min, 72 for 3mins for 30 cycles. Secondly please guide me for pathotyping I am using eae, aggR, vt, It, st, ipaH, virF, daaE?
Which paire of primers are using for the 16S rRNA gene amplification?
Two possibilities here.
(1) Your E. coli DNA has become degraded - in which case the phylotyping PCR will not work either.
(2) Your 16s primers do not work. Have you run a control with a sample which you have successfully amplified before?
Thanks all for your time, we have cultured it again by re steak and it seems there is some contamination, so isolating the culture again hope it works. As we also ran the control samples with 16S and it amplified the product, it means that 16S works but problem in culture.
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