Adaptors for join SalI to BglII

frank rozsa at
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

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!

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

email: rozsa at

"You're a better man than me doc, but that's because I'm a rabbit"

                                   -Bugs Bunny


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