NuSieve Agarose

[Mike Morris]

In article AEv at news.otago.ac.nz, biocmag at sanger.otago.ac.nz (Michael Gieseg) writes:
>>
>>I'm currently trying to sub-clone a 300bp fragment using NuSieve agarose.  Now
>>I know that in-gel-ligations can be performed in NuSieve agarose without much
>>difficulty but I was wondering if it was also possible to endfill the StyI ends
>>(5' overhang) of the fragment in the gel (with klenow) before carrying out the
>>ligation?  Has anybody tried this?

  I haven't tried it; but agarose inhibits DNA Pol 1 activity, so may
  well do the same to Klenow.

>>One thing that does bother me is that to heat-inactivate Klenow you have to
>>heat it for 10 minutes at 75 degrees celius.  However the NuSieve protocol for
>>in gel-ligations says don't heat the mixture over 70 degrees celius.  What harm
>>would heating it over 70 degrees celius do or can the heat inactivation step be
>>missed out?

  1/ Does Klenow really survive at 60°C? I'm surprised...
  2/ You don't want to overheat, because if your fragment starts to denature 
     it might become difficult to ligate ;)

Mike

*******************************************************************
Michael Morris PhD            
Division of Medical Genetics       tel (Switzerland) (22) 702.56.94
CMU, University of Geneva          fax (Switzerland) (22) 702.57.06
Geneva, Switzerland                email mike at medsun.unige.ch