HELP ME!! (lig/trans: UV and CIP) -- repeat message

[Joel A. Kreps]

hmmoss at MAIL.MED.CORNELL.EDU (Heidi Moss) wrote:
>
> 
> I am back again (the "HELP ME!!!" person) looking for more info 
> regarding my construct troubles.  
> Many thanks to the numerous 'netters who have given me such helpful advice.
> (:
> After performing every control experiment in the book, here are some 
> final questions and conclusions:
>  
> 1) Our lab owns only a 254nm UV lamp/photo apparatus. Obviously, long 
> wave is better for viewing/cutting-out my inserts. How grave is 
> short-wave UV exposure in terms of DNA damage? How long of an exposure can 
> cause trouble? What are my alternatives at this point? Could THIS be my 
> major source of difficulty? I do take time to carefully cut out my inserts 
> since I want a small clone from a large, multi-banded lambda DNA.
> -- Based simply on transformation plate appearance, the DNA (both or 
> either vector/insert) that was exposed to the UV DRASTICALLY reduced 
> transformation efficiency.
> -- "Shotgunning" plain, non-UV/genecleaned/dephosph. digested DNA worked 
> best -- however, I now have to sift through every possible construct to 
> find the one I want!!!
> 
> 2) Dephosphorylated vector (5ug/100ul vol, 3U enzyme: 30' 37 degrees, 30' 55 
> degrees, 10' inactivation + EDTA at 65 degrees, phenol CIAA, etoh, geneclean)
> + genecleaned insert REALLY gave me problems: I obtained ZERO colonies after 
> transformation, and my ligation on an agarose gel showed that the vector 
> size/amount remained the same as non-ligated: meaning that DEPHOSPHORYLATED 
> VECTOR WAS UNLIGATABLE. COULD SOMEONE EXPLAIN THIS? IT'S NEW BOE.MANN. AP 
> ENZYME!!What should I do? At least I have the benefit of blue-white selection. 
> Others in the lab are not as fortunate...
> 
> ANY VIABLE ALTERNATIVES, SUGGESTIONS, COMMENTS ARE WELCOME.
> Many thanks again!!
> -- Heidi
> 
Dear Heidi,
Your phosphatase may have chewed your DNA ends up so that they could
not be ligated.  BMB phosphatase has done this before.  I use shrimp
alkaline phosphatase from USB and follow the directions completely, it has v
worked very well for me, you may want to try it.

As usual, I have no affiliation with USB.

Good luck,

Joel A. Kreps
Dept. of Biochem. and Mol. Bio.
UMass at Amherst, MA