Library Multimerizing After PCR

yuan.li · 04-19-2010, 06:43 PM

This is the most weird thing. I wonder if anyone has seen this problem:
On Agilent DNA 1000 chip, my paired end library after PCR amplification has a little bit of high molecular weigh stuff that looks like multimers of the fragment of interest (~300bp). So I purified it to remove the high MW stuff and re-ran it on bioanalyzer but again I saw that same multimer!

Any idea what it is? I thought it was a PCR artifact initially. If that was the case it should be gone after the purification step but it's not. Is it a real multimer?

sulfobus · 04-20-2010, 04:43 AM

It could be heteroduplexes forming during late stage PCR or during purification (esp. using chaotropic detergents s.a. guanidine salts in combination with heat). They migrate slower on gel, check section II in Kanagawa T (2003).

Edit: This should only be prominent if you have homologous regions in your sequences. But maybe the adapter regions are long enough to bring non-identical DNA strands together.

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04-19-2010, 06:43 PM	#1
yuan.li Junior Member Location: CA Join Date: Oct 2009 Posts: 2	Library Multimerizing After PCR This is the most weird thing. I wonder if anyone has seen this problem: On Agilent DNA 1000 chip, my paired end library after PCR amplification has a little bit of high molecular weigh stuff that looks like multimers of the fragment of interest (~300bp). So I purified it to remove the high MW stuff and re-ran it on bioanalyzer but again I saw that same multimer! Any idea what it is? I thought it was a PCR artifact initially. If that was the case it should be gone after the purification step but it's not. Is it a real multimer?

04-20-2010, 04:43 AM	#2
sulfobus Member Location: Denmark Join Date: Oct 2009 Posts: 12	It could be heteroduplexes forming during late stage PCR or during purification (esp. using chaotropic detergents s.a. guanidine salts in combination with heat). They migrate slower on gel, check section II in Kanagawa T (2003). Edit: This should only be prominent if you have homologous regions in your sequences. But maybe the adapter regions are long enough to bring non-identical DNA strands together. Last edited by sulfobus; 04-20-2010 at 04:50 AM.