linker annealing and clean-up

Anthony Tomlinson tomlinson at pplros.demon.co.uk
Thu Nov 23 10:16:09 EST 1995


In article <Pine.A32.3.91.951123110703.34859D-100000 at spider.usp.br>
Francisco G da Nobrega ib - bio 7588, fgdnobre at usp.br writes:
>Is anyone familiar with the annealing conditions for 8-mers that=20
>constitute a linker?=20
>How one is supposed to clean-up the linkers before=20
>use in ligation reactions?
>How much to use in liagtion?=20
>Will excess linkers inhibit the reaction?
>Thanks for the feedback!

Provided your oligos are clean (HPLC is best) just heat to 80°C and cool
to
RT over 20' or so, in 1x ligation buffer, or PNK buffer, or whatever. Use
at
equimolar levels in the ligation as you would for a normal insert.  I've
never "cleaned up" linkers before ligating, and
never had any problems.


Cheers

Anthony.



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