help: c oning PFU pcr fragment

Richard P. Grant see_sig at
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.



Richard P. Grant MA DPhil     |             rgrant at
Senior R&D Scientist          |             work:
Cambridge Molecular           |      home:           
 -- 'Practising biochemistry without a licence' - E. Chargaff --

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