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homemade T addition or blut PCR cloning?

pgegen at rnaworld.bio.ukans.edu pgegen at rnaworld.bio.ukans.edu
Tue Dec 5 19:22:06 EST 1995

In <49qi9e$d46 at epx.cis.umn.edu>, shin at biosci.cbs.umn.edu (Shin Enomoto) writes:
>I want to clone a PCR fragment into a expression vector I made.  The 
>polylinker has a Pvu II site and need to make a in-frame fusion into this 
>1) Clone the PCR fragment as blunt by using a polymerase that does not 
>give over-hangs
>2) Clone the PCR fragment by first polishing the extra base and blunt clone
>3) Add a extra T to the pvu II site either by taq polymerase or terminal 
>deoxy transferase and clone in a PCR fragment with an A over-hang.
>4) Design a restriction site into the PCR primers to clone
>Which one of the above is likely to be the easiest? I will summarize the 
>Thank you

Use VENT polymerase, or another proof-reading polymerase. We use it routinely and get good blunt-end ligation. Second-best would be to add cloning sites to the primers. The other two approaches are far more cumbersome than you need. 

|  Dr. Peter Gegenheimer         | voice: 913-864-3939    FAX:913-864-5321  |
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