Altered Sites II

Shahram Mori smori at nmsu.edu
Sun Nov 26 19:03:08 EST 1995


Don Haut (c601591 at mizzou1.missouri.edu) wrote:
: I am having considerable trouble with the Altered Sites II system from
: Promega.  My problems are centered around the R408 helper phage.  ALL of
: the colonies that I get after using the system are contaminated with the
: R408 helper phage and it is impossible for me to use any of my mutants. 
: When I try to grow up the colonies for sequencing or cloning I get an over
: growth of R408 which makes the plasmid produce ssDNA instead of clonable
: DNA.  I was wondering if anyone else had experience with this kit and if
: so have you had any problems with it.

: Thanks in advance,

Don,
Technically this shouldn't be possible. R408 has a muation (deletion) in 
its DNA involving packaging. It delivers the proteins for packaging 
but can not package its own DNA. So you are supposed to get predominantly 
your ss pALTER DNA packaged.

If the DNA that is packaged is R408's, then there must be something wrong 
in the stock that Promega sent you. I think Promega sends you another 
Phage an M13, I believe. Use that. It's supposed to have a lower yield 
than 408 with JM109, but that'll at least tell you if your 408 is ok.

ALternatively call Promega and tell'em their phage sucks.
good luck.


: Don

: Don Haut 
: Molecular Microbiology and Immunology
: University of Missouri-Columbia
: C601591 at showme.missouri.edu
: 314-882-3171

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Shahram Mori
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New Mexico State University
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