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Old 07-28-2016, 01:48 PM   #7
ECO
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Location: SF Bay Area, CA, USA

Join Date: Oct 2007
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IME, Klenow Exo- is unnecessary and only was ever included because the very very first library kits from Solexa had it in there. T4 pol is a monster at end removal and fill in. The blunting phase of the reaction is over in <10 minutes (at least with clean DNA)

The best combination if you really want a one-pot reaction is T4 PNK + T4 Pol + Taq + dNTP. 20C is the ER phase. 65C is the heat-kill + Taq tailing phase. Non-templated addition is better at 65C than at 72C.

You WILL get lower A-tailing efficiency with Taq in the presence of all 4 nucleotides, but it's not that bad and probably worth the time savings.
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