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End probes/Plasmid rescue

Terrence P. Delaney tpd4 at cornell.edu
Fri Aug 30 17:12:53 EST 1996


In article <506i5q$slf at mserv1.dl.ac.uk>, gbcb42 at udcf.gla.ac.uk (Carol
Johnstone) wrote:

> This may be a niave question but I am new to using YACs :-
> 
> I plan to isolate left end probes from YACS from both the EG and EW
libraries. 
> The method I have is straight forward but the first step involves
digesting the 
> yeast DNA with XhoI, NdeI or XhoI + SalI. Does the choice of enzyme depend on 
> which library the YAC is from or what?
> 
> Thanks for the help
> 
> Carol


Carol, 

The library constructed by Ward and Jen is in pYAC3, while the Grill and
Somerville library was made in pYAC41.  

The latter vector is derived from pYAC3 by:

1. Introduction of an EcoRI cloning site into the SnB1 cloning site in
pYAC3 to produce pYAC4; 

2. Introduction of an:
<EcoRI><NotI><T3 prom><BamHI cloning site><T3 prom><NotI><EcoRI>
fragment into the EcoRI site of pYAC4 to produce pYAC41.

Aside from the above described manipulation of the cloning site, and
ablation of a few EcoRI sites in the making of pYAC4, the two vectors are
the same.  Therefore you should not have to use different enzymes for your
left end isolation experiments (via left end plasmid-rescue).  

The Ward/Jen library was made by randomly shearing genomic DNA while the
Grill/Somerville library was made by partial BamHI digest.  Therefore only
the Grill clones can be released with the cloning enzyme (BamHI).  By the
way, the vector contributes about 5.5 kb to the final clones obtained.

Inverse PCR can also be useful to isolate end fragments.  In this case,
the primers used do vary according to the pYAC vector used.  However the
enzymes used for iPCR can be the same.

Good luck,

Terry Delaney

  Terrence P. Delaney                 
  Cornell University   
  Dept. Plant Pathology
  Ithaca, NY 14853-4203            
  tpd4 at cornell.edu
  http://ppathw3.cals.cornell.edu/ppath/delaney/delaney.html



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