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Old 01-19-2010, 08:09 AM   #2
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Location: Oklahoma

Join Date: Sep 2009
Posts: 411

1. Technically there will be a shift upwards in after A tailing, but unless you're running a gel with 1bp resolution you shouldn't notice a shift at all. Especially a normal agarose gel. Heck, even the bioanalyzer can be plus or minus 5-10bp.
2. Adapter dimers should be less than 100bp in length, given that the adapters themselves are ~33bp in length. These should be mostly taken care of by the minelute columns, which I believe have a size exclusion limit of ~75bp.
3. After ligation you should see your band shift upwards ~50bp. If you don't see a band shift you might not have properly ligated adapters.
4. For size selection yields the Qiagen columns run ~80% yield each, when everything works right. Agencourts SPRI beads are typically higher, ~90+%, but take a lot longer to run. Illumina has some new library tech coming down the pipe which should remove several purification steps to help increase overall yield.
5. 18 cycles for PCR seems a bit high. Are you worried about PCR artifacts? You could probably get away with 10 or less. You only need ~2ul of a 10nM library in the end.
6. Going back to your shearing, are you really seeing genomic DNA after Covaris shearing?
7. Do you have access to a bioanalyzer? They're really helpful for this sort of thing.
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