cloning small linker fragments

Mikhail Alexeyev malexeye at JAGUAR1.USOUTHAL.EDU
Wed Dec 3 20:25:20 EST 1997


Clemens Kittinger wrote:

> I=B4ve a small linker fragment ( 51 bp ) I want to clone in a 4,2 kb larg=
> e
> vector. Sorrowly I=B4ve to clone it Xba I - Xba I. I=B4ve played a lot on=
>
> the ratio vector vs insert, but I didn=B4t get clones until now.
>
> If you have experience in cloning such small fragments, please answer
> me.
>

I just did some last month.  One was 50 bp polylinker in 2.6 kb vector
(blunt-ended).  Just anneal two unphosphorylated oligos in ligase buffer
and ligate a bunch of insert (2-10ug) with 0.2 mg of vector.  I did that
in the presence of restriction enzyme (PvuII) that I've cut vector
with.  Ca. 80% cof clones had insert.  In a parallel experiment, I
"kinased" linkers prior to ligation (ca. 50% insert).

Same strategy worked for two other pairs of annealed oligos (15 bp).

M.A.




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