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Old 06-04-2013, 07:04 AM   #4
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 Phillip,

[...]

I also suspect a contaminant in the DNA, and as or DNA is normally prepared in to TE buffer I have done a phenol DNA prep and resuspended an ethonol washed DNA pellet in the Illumina buffer with RNAase A, fragmented, ampure xp clean up and resuspended in Illumina RSB buffer, but it still failled.

I'll have a look into the Zymo kit thanks. Would you do this and the RNAse step post fragment?

Thanks for the input
Adam
We try to get the customer to do it before bringing us the sample. RNAse first, to degrade RNA small enough that the genomic DNA purification column will exclude the degraded RNA oligos. Then the column -- hopefully removing any last traces of small molecule contaminants (eg, phenol, maybe even cesium if you are getting DNA from an "old school" researcher) or even DNAses. Then we would sonicate.

Then we still do the Ampure prior to end polishing.

One thing to note here is that the pre-clustering PCR amplification gave you a chance to remove from consideration "damaged" amplicons. This would include amplicons with inserts containing non-amplifiable sections. (Due to abasic sites, pyrimidine dimers, etc.)

So if you think about that it becomes a little scary. Before you had this pre-clustering PCR step to shield clustering from weird epigenetic modifications of the source DNA. How robust is clustering to these modifications? If it is more robust, then we are better off than we were before. If it is worse, then there could be issues. Worse, the issues would be sample-specific.

For us it has been "so far, so good". We were getting good results with the pre-amp kit and then seemed to get good results with the no-amp kit. Not a big problem, really. If you decide you really want the amp kit, you can just buy the nano kit. Or the old TruSeq DNA kit until December.

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Phillip
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