I have a problem with the PCR amplification of nickel& cobalt resistance genes. The genes are not getting amplified under normal PCR mastermix and temperature conditions. Is there anything that I have to concentrate very specifically in getting the gene amplified?
Check if your DNA is pure or not. If it is not pure, process it and then do PCR. some impurities may interfere your PCR reaction. and also check whether the primer is specific to that gene or it is universal or not. if it is specific for a particular group of organisms get a universal primer and try.
Thank you for the valuable suggestions. The DNA that I am using should be fine as I used it for the 16S identification. The primers are very specific for the gene of interest and is not specific for particular group of organisms.
Check the MgCl2 concentration in the master mix. and also check the melting temperatures of the primers, and do PCR according to that.
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