Why 1.5 kbp PCR amplified gene on sequencing gave only forward (500 bp) and reverse (512 bp) sequences?

06 June 2019 1 7K Report

I have PCR amplified 16S rDNA (1.5kb) from a symbiotic bacterium with universal primers and the amplifird 1.5kb fragment send for sequencing . The forward (500 bp) and reverse (512 bp) sequences were obtained as sequence result. I would like to know why the sequence result of central portion of DNA didn’t come up?

Craig Silver

I strongly suspect that the reason you are only amplifying short portions close to the primers is due to too short of an extension time. We use the approximate rate of 1min per 1kb of amplification in our lab, with great ease. So for a 1.5 kbp product you likely need 1:30 at a minimum of extension duration. Check your cycling conditions.

Good luck!

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