poor cloning efficiency w/Pfu turbo

Wolfgang Schechinger marsupilamiwolfgang at web.de
Wed Jul 31 08:43:24 EST 2002


Paul, 

taken in account that Taq survives the prec, as annotated by someone, and
you have a long incubation time for Sfi I at 60 or 65 degC, you might
indeed get some fill in.

You could check if your primers also will be cut by Ngo MIV (sometimes also
named NgoMI), you may get it from e.g. NEB. Cuts nicely without requiring
either 60 degC nor a long time and may be used as a substitute for SfiI in
some cases.

Wo

At 05:57 31.07.02 GMT, you 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.


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