Help - cloning into dephosphorylated plasmid vector

"Alexander Kraev" bckraev at aeolus.ethz.ch bckraev at wawona
Thu May 25 12:28:57 EST 1995


In article <3ptrfk$g08 at woodstock.socs.uts.EDU.AU>, Nick dos Remedios <N.dosRemedios at uts.edu.au> writes:
>In a nutshell: I am unable to ligate foreign DNA into the ECORI site of
>my plasid vector (pcDNAI) after the vector cut with EcoRI then treated
>with C.I. alkaline phosphatase. Cut vector re-ligates back together quite
>efficiently, therefore the problem seems to be the CIAP step but I have
>been unable to fix it [I *think* my overhangs are being eaten by the
>CIAP].
>
Although I have been using Boehringer CIAP, it is quite unlikely that
your enzyme is of such low quality, that it chops DNA ends. I suggest
that instead of trying a "test insert", you repeat your experiment
with ligase, using a EcoRI digest of any DNA, but just heat inactivate
the restriction enzyme. You may also try the following controls:
1. 1 ng of your vector, untreated - >1000 colonies upon transformation.
2. 1 ng of your vector, treated with EcoRI, heat treated and self ligated,
should give 300-500 colonies.
3. 1 ng of the same vector, as in 2. but treated with CIAP and self-ligated,
should give near zero colonies.
When your vector does not ligate, it may have damaged ends, or just as
well something inhibiting ligase, coming from the procedure of removing
CIAP.
I routinely do not inactivate CIAP, just add dye to the reaction and
gel purify the vector. It works like a charm!
( No connection to Boehringer )
Hope this helps

*************************************************************************
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



More information about the Methods mailing list