Hi! I am doing reverse transcription using Superscript II and anchored oligo(dT), and making the second strand of cDNA using RNaseH and DNA Pol I. The anchored oligo(dT) has twenty Ts, but when I sequence the cDNA, I noticed that I don't have all twenty Ts and some will not have any. I am losing the Ts during the second strand step, not the first strand step. When I replace one of the phosphate bonds in the oligo(dT) primer with a phosphorothioate bond, the remaining Ts become longer suggesting that some exonuclease activity might be the cause. E coli DNA Pol I has 5'-3' exonuclease activity and I think this might be eating the Ts in the primer. Do you know what might be causing this and how to prevent it? Thank you.
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