PCR of lambda gt10

Steven Sullivan sullivan at gwis2.circ.gwu.edu
Wed Aug 2 19:14:28 EST 1995


mcelverja at phibred.com wrote:
: 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


How do you know you're not missing some large inserts, due to the size 
limits of normal PCR?





More information about the Methods mailing list