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Dear all, I performed sequence capture of target exons using NimbleGen SeqCap EZ strategy. Now I have to proceed to emPCR (Lib L). Should I denature my for 95 C degrees for 2 minutes?
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No you don't, after LM-PCR only 1 of the strands will be able to get PCRed on the bead. -
Ok, thank you Zaag. May I ask you how many copy per bead do you use in emPCR lib L? I would start trying 1 cpb since it is the first time that I perform this emPCR. Do you have any suggestion?Comment
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After sequence capture I usually end up between 1 and 2 cpb; so if you measured your sample with a Qubit before calculating the nr of molecules my guess would be that 1cpb would be a safe number.
Usually I do one SVE with 1 cpb and one with 2 cpb and after calculating enrichment I choose a cpb for my bulk emulsion.Comment
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Can I ask you what is SVE?Comment
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That would be a small volume emulsion. They come in three sizes, small medium and large.Comment
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Ok.Thank you very much for your reply. I was wondering if you also perform SeqCap EZ Choice...I have a lot of question regarding this method but I don't think many people of this forum perform SeqCap and then run on a GS Junior platformComment
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Yeah we do that but on the 454-FLX+ so no junior here. I am not sure what exactly are the differences regarding emPCR and such, but if you have questions just mail me, or ask them here (mail is faster answer).Comment
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