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Old 01-09-2018, 04:22 PM   #29
HiroMishima
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Location: Nagasaki, Japan

Join Date: Aug 2009
Posts: 15
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Thank you for your question.

Quote:
Originally Posted by korostin View Post
Thanks for links to your article!

1. As I understand from your article you decided to avoid cuttting ampliseq primers. Why?
2. Why you used uracil depletion technique instead of U-passing with Phusion U from Thermo with average dNTP's? In total U-containing part of library after PCR would be tiny (and would not form clusters on flowcell)
A1) We did AmpliSeq primer cutting. For amplicons, we used uracil DNA glycosylase and endonuclease IV. This step obtained amplicons without primers outside innermost uracil.

A2) Because we wanted to use regular polymerases already used in related protocols. This may be important for researchers using other protocol hacks . I agee with you. Uracil-tolerant polymerase may work.

Hiro.
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