Cloning Pfu fragments...help|

[Garry Myers]

Hi Lorenzo,

I used Pfu for the last 8 or 9 months and it worked a treat in PCR; v. clean
reactions etc.  However, I also wanted to clone these Pfu-amplified
fragments; try as I might, I could not clone a damn thing!  I tried every
cloning trick I could find - but to no avail.  I switched back to Taq
polymerase and tried a T-tailing method and also blunt-ending with T4
polymerase.  Both these approaches worked first time.  You couldn't really
make any conclusions about "clonability" of Pfu-fragments from this result,
but it did make me wonder . .

It would be interesting to hear of any successful cloning of
Pfu-amplified fragments.  Surely it is possible?

Cheers,

Garry Myers
Menzies School of Health Research
N.T. Australia
garry at menzies.su.edu.au
 
> Hi net,
>  
> does anybody have experience with cloning of Pfu-amplified sequences?
> We tried to use standard blunt-end cloning and Klenow filling of the fragments
> but nothing seems to work...Any suggestions or informations will be greatly app
> reciated. Thanks in advance.
>  
> Lorenzo Frigerio
> Istituto di Genetica Vegetale
> UCSC Piacenza-Italy
> genetica at ipcucsc.bitnet
> botanica at imicilea.cilea.it
> 
>