PCR of Lambda Inserts Question

bugg at mbcf.stjude.org bugg at mbcf.stjude.org
Thu Jul 9 14:33:05 EST 1992


In article <15122 at ucdavis.ucdavis.edu>, fzhedric at hamlet.ucdavis.edu writes:
> 
> Hello,
> 	I am new to this newsgroup so please excuse my ignorance if this topic
> has been covered before.  But I have gotten a number of positive clones in
> lambda gt11 and normally would isolate the inserts by restriction digests of
> lambda DNA preps.  A friend of mine told me that the following would work:
...(alot of protocols for PCR with lambda primers)...
> Now my questions are:  What are the potential reasons why the first reaction mix
> did not work?  Why would one method require Mg and the other not?  What
> would Tween 20 in the second reaction be necessary for?  Could it be for
> dissolution of the viral coat?   If anybody has a standard protocol for 
> amplification out of lambda gt11 I really would be interested to see it.

I don't have any real answers as to why what you did was unsuccessful, except
that I wouldn't put my phage in SM (it never worked for me) and you don't have
any freeze-thaw steps to release the DNA from the phage head.  But, I did want
to say that I do this all of the time by touching a plaque with a sterile
needle and then swishing the needle around in water.  Freeze and thaw the water
with phage 2X and then use to PCR.  This is basically the protocol from
Clontech - I also get the lambda primers from them.  If you look in their
current catalog, page 111, you will see the cDNA insert screening amplimers and
a brief description of the protocol.  There is nothing different from a
standard PCR, except you must freeze-thaw first!

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Barbara Bugg, M.S.  bugg at mbcf.stjude.org       "I refuse to have a battle of
St. Jude Children's Research Hospital          wits with an unarmed person."
Memphis,  TN  USA
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