PCR of lambda gt10

mcelverja at phibred.com mcelverja at phibred.com
Wed Aug 2 08:00:15 EST 1995


stardog at u.washington.edu (Kevin Lease) wrote:
>
> Could someone please send me a good protocol for PCR with a lambda gt10
> clone as my template DNA?  I am particulary interested in hearing how
> people begin their PCR to denature the capsid.
> 
> Thanks,
>         Kevin Lease
> 
Kevin,
  I simply add 1 ul of phage in SM.  I denature normally, ie, 3-4
minutes at denaturing temperature.  The minimal amount of Mg in SM
hasn't mattered to me so far (I use 1.5 mM Mg in a commercial buffer)
and the gelatin was used in early buffers anyway.  I then follow
cycling conditions appropriate for the oligo set in use and the
size of the amplimer.  My rxn volumes are either 50 or 100 ul.
  I've used this technique to amplify clones from library stocks and
to score primary picks (it cuts down on the number of secondary
lifts that you need to do).  When I'm stuck using a library that
doesn't have an easy plasmid pop-out method, I'll also clone inserts 
this way, it's easier than a lambda DNA prep!
  One caveat is avoid the chloroform until after you're done with PCR,
or you'll have to wait a few weeks!
John McElver
mcelverja at phibred.com



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