help: c oning PFU pcr fragment

[Frederik Boernke]

Mr. G. Morley wrote:
> 
> Hi all,
> 
>                 I am having trouble cloning a some PFU pcr generated
> 
> products. This enzyme should produce a blunt ended fragment which
> 
> I have kinased. However it repeatedly fails to clone into a blunt
> 
> ended vector that has been CIP treated. Any suggestions? the fragments
> 
> are purified after kinase treatment by removal from LMP gel. Do the
> 
> fragments require phenoling?
> 
> Any suggestions would be appreciated as it is driving me nuts.
> 
> Thanks in advance.
> 
> Gary morley
> 
> g.morley at ucl.ac.uk

Hi,

why kinase the fragment and CIP the vector? Just leave it as it is,
works
fine in my hands with invitrogen's PCR-blunt vector (However, cip-ing of
blunted vectors was never efficient in my hands). This vector has a
lethal marker which keeps background rather low. If you have it once,
you can easily propagate it yourself in XLI-blue in the absence of IPTG
and subsequent digest with StuI for use with blunt ended fragments.
Hope this helps
Ricky


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

Frederik Boernke
Research Group of  Molecular Plant Physiology
Institute for Plant Genetics and Crop Plant Research (IPK)
Corrensstr. 3
06466 Gatersleben
Tel.  039482 -5 321
Fax. 039482 -5 515
http://www.ipk-gatersleben.de