Adaptors for join SalI to BglII

frank rozsa at umich.edu
Tue Aug 7 14:48:10 EST 2001


HI All,

Question about using adaptors to convert restriction sites:
I have a 4.5 kb insert flanked by SalI (5¹) and XbaI (3¹) , the vector has
Bgl2 (5¹) and Xba (3¹).

I have tried Klenow fill-ins several times without success and am now
switching over to using adaptors. I really do not want to introduce errors
via PCR since it took several round to ³fix² my current clone.

My problem is how to get the SalI site to join to the Bgl2 site. I have in
hand non-palindromic  adaptors from NEB, SalI-XmnI (S1135S) and BamHI-XmnI
(S1108S). These should anneal together to provide a bridge with
SalI-compatible and Bgl2-compatible overhangs.

My question ..
Do I anneal these two 16-mers together without phosphorylation?

Should I kinase both adaptors and if so, before or after annealing?

Should I just throw in this annealed adaptor complex with the digested
vector and insert, ligate the whole mess and hope for three molecule
ligation? 

Should I ligate the annealed complex to only the digested vector, clean up
and then ligate with the insert?

I have seen quite a bit of info on adaptors where you digest with one of the
enzymes to make sure there is only one adaptor per end. I realize
concatamers may form but I cannot cut  with BamHI, Bgl2, or XmnI since these
cut within the insert.

Any help is most appreciated!

Cheers,
Frank
-- 
Frank W. Rozsa MPH PhD
University of Michigan
Dept. of Ophthalmology
1000 Wall St
Ann Arbor, MI
48105

email: rozsa at umich.edu
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