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-   -   10kb+ PCR enzyme suggestions / advice (http://www.molecularstation.com/forum/pcr-polymerase-chain-reaction-forum/72891-10kb-pcr-enzyme-suggestions-advice.html)

CWatkins 04-28-2010 05:11 PM

10kb+ PCR enzyme suggestions / advice
 
Hello,
We are about to run a series of PCRs looking to detect polymorphic TEs and would like some advice on possible polymerase enzymes to use.
We would be looking for bands in the 10-15kb range, but wouldnt be opposed to 20kb+ if the enzyme was capable of that.

Not concerned about its proofreading capability as we are just looking to detect an insert for now and not sequence

I have found about a dozen different candidates from about a half dozen companies and have been pricing them out for which would be cost efficient and most reliable

some that I have been looking at are Takara, Pfu Turbo, Accutaq and KlenTaq LA, Combizyme, and on the high end rTth Polyermerase

Takara and Pfu Turbo seemed to be best for what we were looking for, was just curious if anyone had any input as to the pros and cons to either of these or another reasonably priced enzyme.

Thanks for you time and input.

jonoave 04-30-2010 12:57 AM

Re: 10kb+ PCR enzyme suggestions / advice
 
If you're not really focusing on accuracy/proofreading, I'd suggest skipping the Pfu options as they are costlier, slower, and less efficient (harder to amplify). And that includes AccuTaq (from Invitrogen, if I'm not mistaken?).

From a cost perspective, you could do well with a cheaper option of a modified Taq blend (eg., with Pfu) from several cheaper brands like Promega, Eurx etc.

zhanlin9391 05-04-2010 06:44 AM

Re: 10kb+ PCR enzyme suggestions / advice
 
very helpful post.. thanks for sharing..


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