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Old 12-21-2018, 10:33 AM   #12
aengstrom
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Location: sweden

Join Date: Jan 2018
Posts: 4
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Thanks for the answer, appreciated! The reason for me asking is that I am currently using the library prep (and target capture) from Twist Bioscience, exome and custom panel protocol. The current protocol has a combined mix of enzymes for fragmentation, end-repair and a-tailing - incubation at 32 degrees (fragmentation) and 65 degrees (end repair AND a-tailing - this is what a Twist staff claims) - can this be correct? In contrast, for the setup with enzyme mix from Qiagen/Enzymatics, End Repair and A-Tailing are happening at 20C and 65C, respectively. Any comments on this? This is important to us since we use degraded FFPE samples and we want to keep the fragmentation time in the Twist protocol as short as possible, but still do efficient end-repair... so I was then thinking how does it happen at 65 degrees and then a-tailing also at the same time!?
Also that has confused me a bit is the enzyme mix from KAPA/Roche (HyperPlus) - here only a 65 degree incubation is included whereas in KAPA/Roche HyperPrep, both 20 degrees and 65 degrees are included. (I have learned that the enzyme mixes are the same for both kits/protocols.) Conclusion - the DNA is not end-repaired in HyperPlus (even though the protocol states that), only a-tailed (since it is enzymatically fragmented)?
Sorry, lots of questions and thoughts... I'm trying to understand this fully.
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