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Old 08-20-2014, 07:27 AM   #1
hduborjal
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Default PCR directly on DNA binds to Ampure XP

Hi everybody,
During the library preparation it's possible to conserved Ampure beads during the different enzymatic reaction (it's the with beads strategy for improving yield, Fisher et al. Genome Biology 2011).
Nevertheless, after double size selection, the library is always eluted from Ampure before PCR amplification.
I want to know if it is possible to carry out a PCR directly on library not eluated from Ampure beads after size selection ?
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Old 12-01-2014, 12:54 PM   #2
Beatrice
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I have done it..... it worked. I think the 1X PC buffer by itself remove the DNA from the magnetic beads resulting in (1) free DNA and (2) "inert" beads for the PCR reaction.
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Old 12-01-2014, 01:47 PM   #3
cmbetts
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The short answer is that it depends on which PCR polymerase you're using. When doing some previous methods development, I tested about half a dozen polymerases amplifying either directly on the beads or after elution (All made by a previous employer, so I won't mention names). Some polymerases worked equally well under either condition, while others were completely inhibited by the presence of beads. However, elution from the beads is essentially quantitative (I've measured >99% if you're good at getting every last microliter), so it's probably a safer bet not to risk your samples unless you have a strong incentive not to elute.
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