cloning small linker fragments
kang at msvax.mssm.edu
Thu Dec 4 02:16:29 EST 1997
Vladimir Svetlov wrote:
> 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.
> Vladimir Svetlov
> McArdle Lab for Cancer Research
> Dept. Oncology
> 1400 University Ave.
> Madison, WI 53706
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