erase a base

Tanya Osickey tawny at sage.cc.purdue.edu
Mon Mar 14 20:32:19 EST 1994


tai2 at scws14.harvard.edu (Andrew Tai) writes:

>In article <2ll8e2$904 at ysics.physics.sunysb.edu> mhollowa at epo.som.sunysb.edu writes:
>>In article <hotan-080394162111 at pc10.hrfs.uiuc.edu> hotan@@uxa.cso.uiuc.edu (Douglas T.) writes:
>>> Does anyone have any comments on the erase a base system? 
>>
>>Yes.
>>ARRRRRGGGGGGHHHHHH!!!
>>I've only tried it with the phosphorothiolate end protection and the 
>>results were frustrating.  Out of, perhaps, 50 transformants one or two 
>>were of plasmids whose ends had been protected from digestion.  The rest, 
>>far as I could tell, were bi-directional digestions.  I expect that if I 
>>had used a 3' overhang it would have worked much better.
>>
>	Strange that you had such an unfortunate experience. I was lucky
>enough for my deletions to work just fine with the phosphorothioate end
>protection. I did have a few bi-directional digestions, but not many
>at all. BTW not all 3' overhangs work either. I seem to recall that ApaI, for
>example, won't protect against ExoIII digestion.

>	Andrew Tai
>	tai2 at husc.harvard.edu
  
    I have not personally used erase-a-base yet, but it is in common usage
  in my lab.  We have had several problems with the system.  The two most
  prominent:
   1)  Finding appropriate restriction sites for the two digestion spots
       to allow for filling in the one sticky end for protection.  We
       very recently ordered to vectors supposedly specially designed 
       for this system to provide better end choices.
   2)  Uneven digestion rates dependant on sequence; in other words, we
       don't get a good set of deletions, but instead end up with several
       transformants of one length, a gap of about 300 bp, then more 
       transformnts of another length.  The sequence we are working with
       is VERY  GC rich, so this may be part of the problem.

  -Tanya



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