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Old 06-06-2018, 05:58 PM   #36
zany
Junior Member
 
Location: Beijing China

Join Date: Jul 2012
Posts: 3
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Thank you all the above guys, you provided many informations.

I'm also trying to make my own home-made ampliseq libray kit (for Ion torrent and Illumina)

Based on posts above, I think that the most import thing blongs to :
1) a HIFI DNA polumerase that could tolerrent U in primer( template), there are several candidate: OneTaq (not so HIFI),Phusion U Multiplex PCR Master Mix, KAPA2G FAST Multiplex Kits. I wonder which works best.
2) enzymes that digest primer region and make blunt-end (or single A-tailed) amplicons。 There are many options. UDG & Endo IV might be good choice, because UDG has 100% activity in Endo IV buffer, and Endo IV have both endonuclease and 3'-5' exonuclease activity. However, UDG & Endo IV treated end might be still not able to ligate adaptor. T4 PNK might be needed in this case.


Back to the erly beginning, Do we really need to digest primers? Especially in the conditon that so many tricky details underlines.
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