Testing a Rapid Library

[Dynamac]

Does anyone have a protocol and primer sequences for testing a RL by aqueous phase PCR?

Thanks!

Barry

[mps4208]

Not specifically aqueous phase, but maybe the following will be a start.

Matthias Meyer paper in 2008 has the emPCR primer sequences. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2248761/

forward primer 5′-CCATCTCATCCCTGCGTGTC-3′
reverse primer 5′-CCTATCCCCTGTGTGCCTTG-3′

You might also try the answers in this thread http://seqanswers.com/forums/showthread.php?t=4819

[ajthomas]

I made some primers using the sequences found in the TCB (or is it an Application Note?) that describes unidirectional amplicon sequencing using the Lib-L primers. You can use the adapter sequences in there to make some primers that will amplify any library that can be sequenced with Lib-L chemistry.

I have also used those primers to rescue libraries that had various kinds of sequencing problems by amplifying for a few cycles, then size-selecting the product. What you end up with is a sequencing library where all molecules are functional (like an amplicon library) instead of just those that ligated successfully, and that you don't need to denature before setting up emPCR. in my experience, those libraries tend to sequence better than before doing the amplification.

[Dynamac]

Thanks, all.

Presumably, when you reamplify your shotgun library, you're using the same cycling protocol provided in the Small Fragment Removal TCB. I don't suppose the exonuclease step is necessary if you're size selecting again anyway.