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difference in PCR amplicon intensity between standard strain and isolates


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#1 PVB

PVB

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Posted 13 April 2012 - 07:10 PM

Hi all !!

I have been working on isolation and molecular characterization of a Gram negative bacterium. I have been using conventional PCR (with reported primers) for isolates confirmation and characterization. Adding on to it, I have got all my isolates sequenced and confirmed as the organism of my research. The typical problem consistently faced is the amplicon intensity. May it be any reported primer (both genus and species specific) the amplicon intensity of the standard strain is bright enough, whereas, that of all my confirmed (based on sequencing of few housekeeping and other conserved genes) isolates are fainter with two lower weight non-specific bands. I have tried, decreasing MgCl2, primers and dNTPs concentration. But they were of no help. Increase in number of thermal cycling reactions all made no improvement. DNA template of standard strain and all isolates used for PCR are of same concentration. Use of DNA isolated by conventional phenol-chloroform method and kit based all showed zero differences in the mentioned problem. Now, I have got to sequence 2 conserved genes to study the phyologeny of my isolates, and this problem is stopping my work. I have tried to gel purify the specific amplicon to get rid of non-specificity. Unfortunately, PCR with the gel purified product also, resulted in same lesser intensity of amplicon, with same non-specific bands too, with no improvements.

I hope, technical suggestions by PCR experts here, will really help me out.


Regards,
PVB




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