poor cloning efficiency w/Pfu turbo

D.K. dk at no.email.thankstospam.net
Wed Jul 31 10:08:58 EST 2002


pshinn at mail2.sas.upenn.edu (Paul Shinn) wrote:
>   I'm experiencing poor cloning efficiencies when using PCR products 
>generated by Pfu turbo.  When I do the same ligation and transformation 
>using products from Taq (same concentration), I can get 5x more clones.
>   My products have Sfi sites at either end and are further flanked by 6 
>more bases (EcoRI and SalI).  After PCR, they are sodium acetate 
>precipitated and then digested with Sfi overnight.  The next day they are 
>sodium acetate precipitated again and a small aliquot is taken out for 
>ligation with a vector containing Sfi ends.  Then they're chemically 
>transformed.
>   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.

As others mentioned, carryover of Pfu and some nucleotides can be an 
explanation. Another, more likely, IMHO, is that Sfi works/survives
better in Taq buffer than in PfuTurbo. 

In any case, why not use silica-based PCR cleanup (like Qiagen?).
With manifold and money to buy kitsd you can do 24 samples in 
about 5 min. Beats EtOH pption any time and kills/gets rid of Pfu 
100% sure. For ligation, heat killing is usually sufficient, no need to 
pptate or purify. 

DK
 



More information about the Methods mailing list