pGEM-T easy vector gives extra band
Michael L. Sullivan
mlsulliv at facstaff.wisc.edu
Thu Mar 8 10:07:45 EST 2001
There is also a protocal for making T-tailed vectors in Current Protocols
in Molecular Biology. Essentially, you cut your vector with a blunt cutter
like EcoRV. The blunted vector is then incubated at 70 degrees with Taq
and dTTP only, which will add the appropriate single T overhang.
I've used this a sseveral times. I found that I got rather a lot of
background, BUT, if you use a vector with color selection, it is easy to
pick clones with inserts.
I generally don't do this anymore. Rather, since oligos are so inexpensive
now, I just design restriction sites into my primers. But making your own
T-tailed vector is a good alternative if you can't or don't want to do this.
>"R. Jayakumar" wrote:
>> [...] T-tailing it with
>> DNA polymerase and an excess of TTP, which I can very well do in some other
>> vector too. it is this protocol which I wanted.
>Try using the terminal transferase from any vendor, adding dTTP to a blunt
>I don't have the protocol right here, but i think there is a brief one in
>Sambrook, and in some catalogs.
>Hope this helps
Michael L. Sullivan, Ph.D
U.S. Dairy Forage Research Center
1925 Linden Drive West
Madison WI, 53706
(608) 264-5144 Phone
(608) 264-5147 Fax
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