P1 lysogeny: detection and curing

Robert Whittier rfwhittier at hotmail.com
Thu Aug 16 01:58:22 EST 2001


Tip of the hat and thank-you to Dr. Duncan Clark for his apparently
correct diagnosis and helpful suggestion.

Changing to Stabl2 host makes the constructs at least metastable. I had
thought that SURE's mutations in both the recB and recJ genes would knock
out recombination, making it  equivalent to a recA1 strain. The actual
results however are that I obtained target constructs immediately in
STABL2, a recA1 strain, but never obtained a single one in over 100
SURE transformants.

Robert F. Whittier


>>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).



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