cloning small linker fragments

Vladimir Svetlov svetlov at oncology.wisc.edu
Wed Dec 3 15:29:23 EST 1997


In article <3485629A.7F282532 at kfunigraz.ac.at>, Clemens Kittinger
<clemens.kittinger at kfunigraz.ac.at> wrote:

> I´ve a small linker fragment ( 51 bp ) I want to clone in a 4,2 kb large
> vector. Sorrowly I´ve to clone it Xba I - Xba I. I´ve played a lot on
> the ratio vector vs insert, but I didn´t get clones until now.

Phosphatase the cut vector (I routinely use SAP, but CiAP should work too,
just a little bit harder to kill it afterwards). Check the efficiency of
digest/phosphatase treatment by ligating in absence of the insert and with
the zero background ligate a phosphorylated linker (if it's derived by
annealing nucleotides, kinase them prior to annealing and check annealing
on the gel if you think it might be a problem). The ratio of insert to
vector has to be low (to avoid concatemerization), 1:1 to 1:100 usually
works quite nicely. 
Good luck.
Regards,
V.

-- 
Vladimir Svetlov
McArdle Lab for Cancer Research
Dept. Oncology
UW-Madison
1400 University Ave.
Madison, WI 53706



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