Hi all--
I am an old timey AFLP person moving into next gen. I intend to generate PE reads from a reduced representation library of insect DNA, and am worried about my DNA:
1. Genomic DNA was extracted with Qiagen kits, but with no RNase treatment.
2. On agarose gels I see a strong band of intact DNA plus a schmear; at the lowest size range in some samples, I see what I think is an RNA schmear.
3. I have successfully used these samples for AFLP work using capillary electrophoresis. No problems were evident in terms of restriction/ligation/amplification performance.
4. In preparing libraries, I will size select after ligation, and again after PCR if the size range of fragments warrants it.
My question: Given that I will be size selecting, and that restriction/ligation has worked on these samples, is the degraded DNA/presence of RNA going to screw things up? I am worried that my AFLP expertise is going to be totally useless here!
I am an old timey AFLP person moving into next gen. I intend to generate PE reads from a reduced representation library of insect DNA, and am worried about my DNA:
1. Genomic DNA was extracted with Qiagen kits, but with no RNase treatment.
2. On agarose gels I see a strong band of intact DNA plus a schmear; at the lowest size range in some samples, I see what I think is an RNA schmear.
3. I have successfully used these samples for AFLP work using capillary electrophoresis. No problems were evident in terms of restriction/ligation/amplification performance.
4. In preparing libraries, I will size select after ligation, and again after PCR if the size range of fragments warrants it.
My question: Given that I will be size selecting, and that restriction/ligation has worked on these samples, is the degraded DNA/presence of RNA going to screw things up? I am worried that my AFLP expertise is going to be totally useless here!
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