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Old 06-04-2013, 05:07 AM   #2
pmiguel
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Location: Purdue University, West Lafayette, Indiana

Join Date: Aug 2008
Posts: 2,315
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Quote:
Originally Posted by addyblanch View Post
Hi all would appreciate any input on an issue i'm having.

I've moved across to the TruSeq PCR free kit and seem to be struggling with generating a library.

I'm fragmenting by covaris to 550bp in 50ul RSB as described and I've followed the rest of the protocol to the letter, but my best prep is still only 14pM of ligated adaptors (according to Kapa qPCR quantification). I still have approx 400ng of DNA left (quantified by qubit) which i was expecting due to the size selection steps using the beads.

I've asked tech support and they aren't much help.

I've read that sometimes the adaptors need to be denatured before ligation, Illumina replied with "the protocol has been optimised for the conditions published".

Any ideas would be much appreciated

Adam
What?! If you denature your adaptors prior to use they won't work at all. T4 DNA ligase needs double-stranded substrates for ligation.

If you suspect that your stocks have been denatured at some point, then you want to get new adaptor stocks.

You might be able to re-anneal the adaptors, but I don't like your chances as they have already been diluted to a working concentration that will not favor duplex formation without long hybridization times.

The double-stranded portion of the adaptors is not very long -- 10 or 12 bases, if I recall correctly. So even thawing the adapter tube with your hands might be enough to melt them apart.

I always had a suspicion that the old TruSeq PCR kit adapters had some thermo-labile chemistry covalently linking the two strands of the Y-adapters together. Hence their insistence that at least one cycle of amplification be done to "break the fork" prior to clustering.

If that was the case, it will not be with the no-amp kits as no pre-clustering amplification is done.

So if you or anyone else in your lab are used to melting your adapter tubes in a heat block or something, that might be your issue. You really want to thaw on ice.

Past that, I always suspect contaminants in the DNA prep inhibiting the ligation or end polishing reactions. We always do an Ampure post-fragmentation to mitigate this issue. But even that doesn't seem like enough. We now try to get our customers to do a final RNAse treatment on their pure DNA and follow up with a Zymo Genomic DNA Clean & Concentrator column.

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Phillip
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