Sma I religation problem

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