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Polymerase mixture for blunt ended fragments

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#1 Raygoza



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Posted 02 September 2015 - 09:43 AM

Hi, I tried googling and searched the forum but i couldn;t find the right keywords. My question is: can I make a mix of taq and either vent or pfu to obtain blunt ended fragments for ligating them into a PCR ligation vector? 



#2 phage434



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Posted 02 September 2015 - 12:58 PM

I believe any mix which contains Taq will still preferentially add an extra terminal A at the 3' end of amplicons.

#3 bob1


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Posted 02 September 2015 - 12:59 PM

If you want blunt ends, then your best bet is to just use the pfu or vent, you can also make a mix with the Taq if wanted, but be aware that the proof-reading capabilities of the other polymerases means you might lose some of the overhangs generated by taq. If you are using a TA cloning vector and need the overhanging A, then use the vent/pfu to amplify, then purify and incubate with Taq and some dATP at 72 for a few minutes. 

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