Missing two bases of the adapter in the sequenced 5'RACE product ?

Smitha Sivapragasam @Smitha-Sivapragasam

18 November 2016 1 4K Report

I used Ambion First choice 5'RACE kit to analyze the transcriptional start site of my gene of interest in bacteria. I skipped the CIP (Calf intestinal phosphatase) step and followed the manufacturer's protocol from TAP (Tobacco Acid pyrophosphatase) treatment step. I got three distinct PCR products, out of which I sequenced two using gene specific inner primer. Ideally I should have got the sequence of the adapter (reverse complement) followed by the transcriptional start of my gene of interest. But I have one base missing from the adapter sequence for one of the product and two bases missing from the adapter sequence for another product just before the transcriptional start site. Could any of you help me understand what's happening here?

Hebaallaha Hussein

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