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Old 08-04-2009, 10:12 AM   #1
jsandler
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Location: Berkeley, CA

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Default Low Molecular Weight sample and primer dimers

I recently ran a Low Molecular Weight sample on our 454 and got a bad result. Most of the sequences were just primer dimers. Our samples are pooled PCR products, 200-600 bp in length. The LMW protocol says to skip the AMPure size selection/cleanup step after adapter ligation, but this allows primer dimers to be sequenced.
I want to eliminate the primer dimers, but I am afraid that if I do the AMPure cleanup, I will loose part of my sample DNA.
Any ideas about how to eliminate the primer dimers for the LMW sample?
I have attached a plot of the sequence read lengths from the last run.
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File Type: jpg reads_length_TCAG_chart.jpg (13.3 KB, 27 views)
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Old 08-26-2009, 01:27 AM   #2
Susanne
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Hi, if you don't want to use Ampure clean-up or gel extraction (I'm just assuming this would drop out for the same reasons), did you think about something like using a Centricon (or Microcon)? Millipore claims that they cannot only be used for proteins, but also provides DNA cut-off sizes:
Centricon-30 ssDNA cot-off: 60 nt, dsDNA cut-off: 50 bp
I think I have read something like 90% retention. I guess every additional step will not have 100% yield, but is that necessary? If your samples are PCR products, you should have plenty of everything.
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