Blunt end ligation problem

Richard P. Grant see_sig at cmtech.co.delete.uk
Wed Dec 16 10:15:49 EST 1998


In article <757lm3$ck5$1 at nuscc.nus.edu.sg>, scip7290 at leonis.nus.edu.sg
(Low Hai Sim) wrote:

>         I am currently facing a problem with ligating Klenow treated
> insert into a CIP-SmaI vector. 

<deleted>

As someone else has said, CIP sucks.  Shrimp Alk Phos (USB and Roche,
IIRC) work fine.  In my hands, Sma I (from Roche, was Boehringer) works
fine - but you have to watch the conditions.  The trick is to *just* cut
the vector, so you don't get tatty ends.

Digest the vector (I usually use 10 ug, and do the rxn in 200 ul) with
minimal enzyme at 25C, for 1 hour only.  Include BSA to 0.15 mg/ml final
and spermidine to 2 mM final.  Then chloroform extract, EtOH ppt and
separate the entire reaction through an agarose gel.  Purify the cut
vector only.  This should yield nicely blunted ends with minimal
'nibbling', and you've purified it away from uncut vector.

HTH,

Richard

-- 
Richard P. Grant MA DPhil     |             rgrant at cmtech.co.uk
Senior R&D Scientist          |             work: www.cmtech.co.uk
Cambridge Molecular           |      home: www.avnet.co.uk/adastra           
          -- 'Dear Dr Fleming, your lab is a mess' --



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