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


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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