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Old 06-06-2018, 06:06 PM   #37
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Location: Beijing China

Join Date: Jul 2012
Posts: 3

Originally Posted by Buckethead84 View Post
Hi Hiro,

This seems like an interesting approach. Perhaps you could comment on a couple of things to help make me understand the logic behind your approach.

Only the primers contain uracil and in the subsequent amplicons, the opposite strand would be a non-uracil nucleotide. What makes you think the products are blunt after your treatment with UDG & Endo IV? As opposed to being blunted/having the 3' overhangs being chewed away during the first step of the Kapa library prep protocol? I think this is important for those of us trying to put together a homemade protocol since your paper is the first published evidence of getting AmpliSeq on the MiSeq.

Also, what made you decide to go with Endonuclease IV? One of the modifications I've considered for my own protocol is using Endonuclease VIII & UDG together to create 5'-P moieties that allow direct ligation of the amplicons, but I think I would likely need to treat with DNA Pol 1 or T4 DNA Polymerase to actually chew away the 3' overhangs which would be complementary to the AmpliSeq primer sequences and be uracil-less.

I think you are right, when you use Endonuclease VIII, you might need T4 pol(or DNA pol1).When you use Endonuclease IV, you might need T4 PNK.

Then the next question is: How to determine concentration of these enzymes?
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