Blunt lig. help (NL)

Aart A. van Apeldoorn A.A.vanApeldoorn at med.ruu.nl
Fri Sep 22 05:27:05 EST 1995


In article <43sene$qrg at ds2.acs.ucalgary.ca>, johnstoj at acs2.acs.ucalgary.ca
(Jillian Johnston) wrote:

> Are you sure the enzyme is good? Is the concentration of ATP high
> enough?  We aliquot the buffer with ATP because we ahve found ATP
> to degrade after a few freeze/thaws.  Do you add PEG8000 to a
> final conc. of 13%? We use a 40% stock.
> Good luck!
> Jill


Thanks for your suggestions, however i tried bluntligation with and
without PEG8000, but this seems to make no difference the results are the
same. I also use a fresh stock of ATP each ligation so this can't be the
problem. Could it be as the efficiency of blunt ligations are very low,
that you need more DNA than usual for a good transformation ( i'm
transforming into E.coli JM109 and BMH 71 strains), how much DNA do you
need anyway for a good transformation. Instead of blunting my fragments
and plasmid with Klenow, i'm trying it with Pwo DNA polymerase now, this
is a thermostable PCR enzyme wich adds no A to the end as Taq polymerase
does, what's your opinion on this method. I'm looking forward to your
suggestions.

-- 
Aart A. van Apeldoorn
Dept. of Medical Physiology & Sports Medicine
Faculty of Medicine, Utrecht University
MFU-2 "Stratenum"
Universiteitsweg 100
3584 CG Utrecht
The Netherlands
E-mail: A.A.vanApeldoorn at med.ruu.nl



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