poor cloning efficiency w/Pfu turbo

nospam>optushome.com.au "nospam>optushome.com.au" at hgmp.mrc.ac.uk
Sat Aug 17 03:16:27 EST 2002


The reason why is that probably you are using the wrong cloning vector. 
The vector you use for Taq has to have TA overhangs, since those are 
created by Taq. On the other side, Pfu and its modified forms form blunt 
ended amplicons due to Pfu's native 3'endonuclease proofreading 
activity. You should use a blunt ended vector to clone the Pfu amplicon.

Hope this helps.

Regards,

Vladimir Grubor

Roland Hübner wrote:
>>   We can't figure out why the Taq products give more transformants while 
>>the Pfu turbo products will give fewer and sometimes no transformants.  
>>Gel purification of each the products is not an option because we are 
>>cloning hundreds of unique ORFs every month and we don't have the time.
> 
> 
> Hi,
> 
>  do the amplification rxn's look equally clean on gels?
> 
> R.




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