Sma I religation problem

[Martin Osterhoff]

() wrote:

>rpgrant at molbiol.ox.ac.uk (Mad Dan Eccles) wrote:

>>In article <BARRYM.95Jul20125808 at stella.med.utah.edu>, barrym at stella.med.utah.edu (Barry Moore) writes:
>>>>  Should still work, but you need to phosphatase the vector.
>>> 
>>> I would add that you want to be careful with the CIP.  I just spent the last month figuring out that
>>> I was using to much CIP, and it was ruining my cloning (I don't know if this enzyme is so harsh that
>>> it was chewing up my vector ends, or if it was making it through my gel purification to destroy my ligation).
>>> I found that 0.1 U/0.5-1.0 ug vector prevent recircularization.
>>> 

>>Barry,

>>I am _very_ careful with CIP; I never use it. (!)
>>With the usual disclaimers, I find that Shrimp (yes, I said Shrimp) AP from 
>>USB/Amersham is a beaut of an enzyme, and we now use it routinely, even for 
>>blunt ends.  Never had experience with -OH overhangs, but needles to say it's 
>>grreat with -P overhangs.

>>Rgds,

>>Richard

>>-- 
>>Richard P. Grant MA DPhil      rpgrant at molbiol.ox.ac.uk
>>Nuffield Department of Obstetrics and Gynaecology, University of Oxford.
>>http://sable.ox.ac.uk/~lady0266

>>Call yourself a dog? I've seen better hairs on a lavatory brush!

>Hi Barry,

>I had the same problem for a longer time.
>SAP, as well as CIP, has an exonucleaseactivity when used in too great
>amounts. Try to use just a very little drop by only touching the
>AP-solution with your pipette tip. It is normally enough for
>dephosphorylation.

>By, Martin 

I didn't tell my name, please excuse me.
Martin Osterhoff
e-mail: Martin.Osterhoff at rz.ruhr-uni-bochum.de