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Old 07-03-2009, 02:48 PM   #1
seqgirl123
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Default PCR enrichment of libraries in 12 cycles or less?

Has anyone been able to successfully amplify their genomic libraries in 12 cycles or less using the Illumina or NEB reagents for library prep and show successful sequencing?
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Old 07-03-2009, 03:07 PM   #2
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Have you seen this paper? With modifications to the ligated adapters you can avoid PCR completely.

http://www.ncbi.nlm.nih.gov/pubmed/19287394
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Old 07-03-2009, 04:29 PM   #3
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I do not have access to that article. would you be able to upload here or PM it to me?
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Old 07-03-2009, 04:31 PM   #4
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You absolutely do not have a PM with the PDF attached...because that would be wrong.
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Old 07-05-2009, 11:54 AM   #5
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thanks. got the paper
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Old 07-05-2009, 01:45 PM   #6
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You mean, on your own...right??
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Old 07-05-2009, 02:53 PM   #7
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I am wondering why they 5-phosphorylate both adapters in that paper.
The adapter they call A_adapter_b needs it for ligation to the DNA fragment, but the other one ?

Am I missing something for which it might be required ?

seqgirl, I used 10 cycles with 5 ug starting material for paired-end genomic DNA sequencing - no problem.
However, I want to give the PCR-free a try.

Thank you in advance if someone has a hint !
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