subtractive library

[Zhencai Wu]

Hi netters;

I am working on cDNA subtractive hybridization to clone unique genes from differently treated plant tissue using Clontech PCR-select cDNA subtraction kit. I am able to get a few hundred white colonies. I randomly selected 23 clones for sequencing. Among them I got 7 clones with long polyT tail, 5, 2, 2, 2 clones identical. It seems to me that duplication rate is higher than expected. I had another library before without any single clone with polya tail after sequencing more than 60 random clones.

My questions are: Do I expect have many clones with polyA? What rate is reasonable for duplication? I followed the protocol exactly.

Any suggestions and help will be appreciated in advance.

Zhencai Wu
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