P1 lysogeny: detection and curing

Dr. Duncan Clark news at hgmp.mrc.ac.uk
Wed Aug 8 07:15:23 EST 2001


In article <F165w8k69RKLpgTFur20000009b at hotmail.com>, the eminent Robert
Whittier at BIOSCI/MRC Human Genome Mapping Project Resource Centre
wrote
>The fun and games start when I try to insert a rather large piece of
>DNA that contains several more wt and mutant loxP sites. It's a
>directional insertion (Bgl II with BamH I and Sal I with Xho I) so
>it should be a piece of cake. Restriction digests of resultant
>plasmids reveal a menagerie of constructs, many with bands that are
>not stochiometric. I tried transforming the other plasmid with the
>many loxP sites into this strain, and obtained well-defined colones.
>However, when they are picked into liquid media under selection (amp)
>their growth peters out while the cultures are only faintly cloudy.

Why couldn't it be just lethality due to recombination. You must start
with an initial plasmid with lots of repeats and or duplicated sites. I
am then guessing that the loxP site is large enough for recombination to
occur leading to different plasmids.

The only thing I can suggest is to try different E.coli's and or
possibly reduce the plasmid copy no. (different ori needed
unfortunately).

If it is P1 lysogen can you not induce it? What about seeing if you can
PCR a P1 fragment from the E.coli. If there is P1 about it should show
up.

Books with P1 info include Experiments with Gene Fusions and Miller's
classic Experiments in Molecular Genetics 

Duncan
-- 
Homogeneous Fluorescent Reporting Systems for Real-Time Quantitative PCR: 

Optimisation, Probe Technology & Future Systems

4-5 September 2001
King Alfred's College, Winchester, UK 

http://www.dstl.gov.uk/html/specialisations/index.htm




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