Dephosphorylation

Andre Hamel hamel at ccu.umanitoba.ca
Fri Mar 5 12:30:58 EST 1993


In article <1993Mar5.104525.201 at chmeds.ac.nz> cytogen at chmeds.ac.nz (Martin Kennedy) writes:
>In article <C3AB4A.AA6 at ccu.umanitoba.ca>, hamel at ccu.umanitoba.ca (Andre Hamel) writes:
>> In article <9303012249.AA11977 at pelican.dbe.csiro.au> adeno at dbe.csiro.au (both lab) writes:
>>>Hi netters.

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Regarding Martin Kennedy's response:

Sounds good Martin, I'll have to give it a try next time (ie, simply adding
CIAP to the rest. endo.(RE) digestion around 1/2 hour before diluting, etc.
versus the much more laborious ProK/phenol/CHCl3 ext'ns, etc that I
routinely do.

How's the background transformation efficiency for vector-alone ligations?  

Do you simply heat treat for ALL RE digestions? Then simply add this
diluted vector to your ligation?

Cheers

Andre Hamel
Manitoba Vet. Virol.
Winnipeg, Manitoba
CANADA
email: hamel at ccu.umanitoba.ca
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Martin's earlier response:>

>I do all my vector dephosphorylations with Boehringer Mol Biol grade CIAP, by
>adding it into the digestion about 30 - 60 minutes before completion. Then I
>add water to bring the volume of the digestion up to 100ul (I digest 2ug of
>vector every time, thus final conc = 20ng/ul, handy for ligations) and simply
>incubate the whole lot at 75o^ for 15-20 minutes. Thats all.  This seems very
>effective and I've used it for many dozens of different vector preps, and
>generally I get pretty low background vector ligation.  Why bother with the
>prot K and phenol extractions when you really don't need them :-)
>                                                               
>Cheers,
>
>Martin
>
>Martin A Kennedy (E-mail = cytogen at chmeds.ac.nz)    
>Cytogenetic and Molecular Oncology Unit               
>Christchurch School of Medicine                       
>Christchurch, New Zealand                     



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