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Old 03-04-2015, 08:37 PM   #7
nucacidhunter
Jafar Jabbari
 
Location: Melbourne

Join Date: Jan 2013
Posts: 1,231
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Quote:
Originally Posted by SNPsaurus View Post
I would pool the amplicons of each sample and fragment with Nextera using just a 2-3 ng reaction. Then PCR the pools, combine the samples and sequence. So the workflow is:
40 PCRs per sample -> pool the amplicons of a sample -> 40 Nextera reactions total, 1 per sample -> 40 PCRs total, 1 per sample -> pool -> sequence
This approach is the most convenient and less expensive option. Further saving can be made by reducing tagmentation reactions to 1/4 of Illumina recommendation for Nextera XT. In this case PCR volume still need to be 25 or 50 ul to have optimum yield. When using full contents of the kit, PCR can be done with KAPA HiFi polymerase in 25 or 50 ul volume.
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