A short linker
wind at biobase.dk
Sun Oct 26 06:33:04 EST 1997
Yoram Gerchman (yoramg2 at LEONARDO.LS.HUJI.AC.IL) wrote:
: Greetings. I'm tryng to insert a short linker (about 45 nt) into a
: restriction site in the middle of a gene. Now, my idea was to creat the
: linker in such a way that it will leave cohesive ends that fit the RE.
: somthing like
: Anyone tried this?
: Yoram Gerchman
: Division of Microbial & Molecular Ecology
: The Institute of Life Sciences
: Hebrew University of Jerusalem
: Givat Ram, Jerusalem 91904, Israel
: Tel: 972-2-6585175
What you suggest is doable, but look out for the following:
1) Dephosphorylate the vector after cutting to avoid self-ligation.
2) Make sure that your oligoes (the linker) is 5'-phosphorylated. You
can do this yourself or probably make the manufacturer of the oligoes
3) Look out for multiple insertions! Since you use only one RE-site,
you risk that several linkers will be inserted as repeats. Thus,
perform ligations with different vector:insert ratios, the higher
ratio the better the chance of a single linker insertion.
4) To save yourself some sequencing, look carefully for RE's whos
digestion-pattern can reveal if you have multiple insertions.
For example, if RE XxxI cuts on both sides of the site that you
clone your linker into giving a fragment of approx. 500 bp,
you can easily screen your clones with XxxI and identify those
with a single linker inserted (positive clones will then give
a 545 bp band).
5) Your linker may enter the RE site in both directions!
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