TruSeq libraries - double sizing?

jlove · 04-20-2011, 02:28 PM

Has anyone else been getting PCR products that are exactly double the size of the template? I am seeing this occur with the TruSeq kits and also the KAPA enzyme. I have heard some theories - too much input (seems unlikely), samples clumping together, etc.

I'd love to hear if other people have figured out what is causing this problem. It seems to be a new one over here.

Thanks!

pbluescript · 04-21-2011, 05:41 AM

It is due to overcycling your PCRs, so it could also be due to too much input. I have attached an example of two libraries where I tried different numbers of PCR cycles. The gel shows 6-18 cycles in 2-cycle steps from left to right. In this case, I took the 8 cycle libraries.
Here's another thread talking about this issue:
http://seqanswers.com/forums/showthread.php?t=8894

jlove · 04-22-2011, 01:34 PM

Update: it was indeed over-cycling that created the problem. I believe that I was getting the amplicons partially hybridizing at the adapter regions and forming Ys which ran larger on the bioA. 10 cycles (vs 18) completely solved the problem. Thanks pbluescript for the ideas!

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04-20-2011, 02:28 PM	#1
jlove Member Location: Boston, MA Join Date: Apr 2011 Posts: 49	TruSeq libraries - double sizing? Has anyone else been getting PCR products that are exactly double the size of the template? I am seeing this occur with the TruSeq kits and also the KAPA enzyme. I have heard some theories - too much input (seems unlikely), samples clumping together, etc. I'd love to hear if other people have figured out what is causing this problem. It seems to be a new one over here. Thanks!

04-22-2011, 01:34 PM	#3
jlove Member Location: Boston, MA Join Date: Apr 2011 Posts: 49	Update: it was indeed over-cycling that created the problem. I believe that I was getting the amplicons partially hybridizing at the adapter regions and forming Ys which ran larger on the bioA. 10 cycles (vs 18) completely solved the problem. Thanks pbluescript for the ideas!