losing primer sequence during second strand cDNA synthesis

Oh Kyu Yoon via methods%40net.bio.net (by fyzkem from gmail.com)
Tue Jun 22 07:39:50 EST 2010

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.

More information about the Methods mailing list