UV damage, dephosphoryl., and making constructs: MAYBE it's SOLVED???

Heidi Moss hmmoss at MAIL.MED.CORNELL.EDU
Fri Mar 10 14:44:53 EST 1995


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)
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. 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



More information about the Methods mailing list