IUBio Biosequences .. Software .. Molbio soft .. Network News .. FTP

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 
>site.  
>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 
>responses.
>
>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. 

o---------------------------------------------------------------------------o
|  Dr. Peter Gegenheimer         | voice: 913-864-3939    FAX:913-864-5321  |
|  Departments of Biochemistry   | pgegen at kuhub.cc.ukans.edu                |
|    and of Botany               | URL: gopher://rnaworld.bio.ukans.edu:70  |
|                                |                                          |
| University of Kansas           | "When you have excluded the impossible,  |
| 2045 Haworth Hall              |  whatever remains, however improbable,   |
| Lawrence  KS  66045-2106       |  must be the truth."    Sherlock Holmes  |
o________________________________|__________________________________________o





More information about the Methods mailing list

Send comments to us at biosci-help [At] net.bio.net