pfu vs long pcr mix enzyme

Michael Sullivan via methods%40net.bio.net (by mlsulliv from wisc.edu)
Mon Jan 18 15:19:26 EST 2010


My opinion is that you should ALWAYS sequence PCR products if in the  
end it will be important to have it error free (i.e. you are trying  
to express active protein). Even with a high fidelity enzyme, there  
can still be an errors . Sequencing now is a lot easier than doing a  
bunch of experiments only to find out later that you had an error.  
You can't rely on your polymerase having the fidelity to make an  
error free fragment-- it will only reduce the likely hood of having  
an error.

Mike


On Jan 18, 2010, at 9:07 AM, Azam Rahimpour wrote:

> Hi
> I need to amplify a 1.8 kb protein coding  gene for cloning and  
> then expree it in mammalian cells, I used both pfu and long pcr mix  
> enzymes (fermentas). pfu amplification was failed but I had sharp  
> bands with long pcr mix, but I am still uncertain of feidelity of  
> that mix supplier says its error rate is 3 time less than tag, and  
> it means that there may be some errors, I dont know what to do  
> should I do more try with pfu or cloning 1.8 fragment amplified  
> with long mix is safe?
>
> regards
>
>
>
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