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Old 04-19-2010, 06:43 PM   #1
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Location: CA

Join Date: Oct 2009
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Question 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? is offline   Reply With Quote
Old 04-20-2010, 04:43 AM   #2
Location: Denmark

Join Date: Oct 2009
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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.
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