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Old 02-07-2011, 06:28 AM   #1
sarjo
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Question gDNA for Illumina--how pure is pure?

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!
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Old 02-08-2011, 12:12 PM   #2
sarjo
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In hopes of encouraging a reply, I have attached a photo of one of my quantitation gels to show what I'm concerned about.

Any advice would be most welcome!
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Old 02-08-2011, 12:59 PM   #3
cliffbeall
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If it were me, I would probably RNAse (be sure to heat treat the RNAse to get rid of DNAse), Phenol extact, and EtOH ppt. The Illumina adapters and reagents are pretty expensive. But I haven't tried a sample with that much RNA contamination.
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Old 02-09-2011, 09:33 AM   #4
peromhc
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Sarjo, Why not Bioanalyze these samples to get a better handle on RNA/DNA size, quantity, etc...

The gel's like you show would cause me to worry, and unless the samples are unique, redo the extraction.
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Old 02-09-2011, 10:12 AM   #5
Michael.James.Clark
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It will probably be okay, but why not go ahead and RNAse treat those samples? You can just re-do the Qiagen step with RNAse this time. See if that gets rid of the smear.
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Old 02-09-2011, 11:53 AM   #6
sarjo
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Thanks very much guys! I am new to this and having newbie anxiety--having some feedback from experienced NGSers helps enormously.

In regard to your suggestions:

peromhc: I don't have direct access to a bioanalyzer, but I could ask a nearby lab to run some samples for me.

Michael and cliffbeall: Regarding redoing the Qiagen from RNAse onward, I can't seem to find ANY info about whether this will work given that my DNA is in elution buffer now. Do you guys have experience with this?

The samples are unique and represent a few years of selection, crosses, and assays--so I am very scared of compromising them. In some cases I have no more tissue available; in others, re-extraction would be an option.
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Old 02-09-2011, 11:55 AM   #7
Michael.James.Clark
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Yeah, I've done it from the EB. It will work. Just re-extract it from the EB using the same kit.

If you have questions about the Qiagen stuff, they have very good customer support reps on the phone. You might want to give them a call before hand and make sure everything will come out the way you want--just tell them you forgot to do RNAse treatment and ask what you should do.
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Old 02-09-2011, 12:10 PM   #8
sarjo
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Many thanks, Michael. I will do as you suggest. I'm always a bit chary of customer service, as the fix often seems to be "buy this other thing from us," but now I think I know exactly how to frame my question. You are so nice to take the time to help!
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