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Old 01-07-2015, 12:06 PM   #6
microgirl123
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Location: New England

Join Date: Jun 2012
Posts: 200
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We're a core facility so the first round of PCR is up to the investigator. I ask them to perform their initial PCR in a 50-ul volume and run 5 ul on a gel to be sure there's a single band (even if it's faint). The other 45 ul is submitted to us and cleaned up with Ampure XP - I usually elute in only 28 ul to concentrate and run 5 ul of this on a gel to be sure the cleanup didn't go horribly wrong. If there's still a band (even faint), I quantify this with Qubit and add up to 50 ng of DNA to the second reaction, which is also 50 ul.

Depending on how much DNA I was able to add, I run 5-8 cycles of PCR. Then another quick Ampure cleanup, eluting in 25 ul, and running 5 ul on a gel. I've also had better luck with the Phusion HF taq mastermix than with the KAPA taq Illumina calls for.
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