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Old 11-13-2014, 06:30 PM   #6
bunce
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Location: Perth

Join Date: Sep 2012
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Quote:
Originally Posted by cmbetts View Post
As for cross contamination, having separate pre and post PCR benches is critical (much more common in corporate labs), but you shouldn't need to do many PCR cycles at all to add the p5/p7 and indexes, so you really just need to make sure your first PCR product and final libraries stay the hell away from the bench where you set up the first round of PCR.
I would not be so hasty in talking down the contamination risk in amplicon workflows - our ancient DNA set-up is 'extreme' and we still get bleed-though of tags (we never re-use index pairs). If you are doing a 2nd round PCR you still need to open that tube in a post-PCR 'area' to spike in the 1st round PCR. Amplifying amplified DNA that then undergoes another amplification (cluster generation) can cause headaches downstream from chimera formation to contamination that builds over time. But, again, it does depend on the end use of the data. thats my 2cents worth - cheers, Mike
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