help: c oning PFU pcr fragment

Richard P. Grant see_sig at cmtech.co.delete.uk
Tue Sep 29 10:32:30 EST 1998


Hi Gary

I would guess that your gel-purified fragments are not as pure as you
would like.  This is a common problem with agarose gel purification and
ligations.  You might try a different method of gel extraction, or
column-purification or ammonium acetate precipitation.

Which enzyme did you use to blunt the vector?  Some common blunters can
give trouble.

Using CIP, it *is* necessary to clean up by phenol-chloroform.  Follow
this with chloroform and then a EtOH ppt, and you don't need to
gel-purify. OTOH, shrimp alk phos from USB inactivates at 70C and obviates
the need for organics.


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           
 -- 'Practising biochemistry without a licence' - E. Chargaff --



More information about the Methods mailing list