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

"Alexander Kraev" bckraev at aeolus.ethz.ch bckraev at wawona
Mon Mar 13 04:55:13 EST 1995


In article <Pine.AUX.3.91.950310142402.18053B-100000 at mail.med.cornell.edu>, hmmoss at MAIL.MED.CORNELL.EDU (Heidi Moss) writes:
>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. 

At your earliest possibility, buy a long-wave UV source. The hand-held versions
are not expensive. If it is not possible, avoid cutting fragments from gels.
In fact, many cloning experiments are done easily without this. In many
instances it pays to learn to screen 20-40 clones than working towards a
perfect cloning, where you would have to analyse only 3-6 clones.
>
>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!

This is exactly what dephosphorilation is for! The vector should NOT be
self-ligatable. In principle it is possible to damage the ends during this
procedure, but today's enzyme ( particularly the one you mention ) quality
is very high, so it is rather unlikely. Try to add to your vector any digest
with compatible ends during ligation, and the number of colonies should jump
up dramatically.
Cheers,

>ANY VIABLE ALTERNATIVES, SUGGESTIONS, COMMENTS ARE WELCOME.
>Many thanks again!!
>-- Heidi
*************************************************************************
Alexander Kraev, Ph.D.                 Internet: bckraev at aeolus.ethz.ch
Lab. of Biochemistry III               Phone: 0041-1-632-31-47
Swiss Federal Institute of Technology  FAX:   0041-1-632-12-13
Universitaetstr.16
CH-8092 Zurich
"Some ideas are obscure not because they are complex, but because they 
 are excluded from our circle of comprehension" - Kozma Prutkov



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