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ProofReading Enzymes help!

ProofReading Enzymes help! - PCR - Polymerase Chain Reaction Forum

ProofReading Enzymes help! - PCR - Polymerase Chain Reaction Forum. Discuss and ask questions about PCR troubleshooting, PCR protocols and methods, PCR products, and PCR theory.


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Old 11-25-2010, 09:51 AM
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Default ProofReading Enzymes help!



I am trying to amplify a 1.8kb insert from a clone, using Pfu. I have standardized the conditions by using Taq. i used a graditent from 50-60 degrees, and i got the desired amplicon for all temperatures. the weird part is reaction just does not seem to work with pfu. i have tried changing template concentration from 1 pg-500g, i have varied MgCl2 concentration as well, and i have also tried using a gradient. i am completely and totally out of clue as to what might be going on. i have also tried Vent and Deep Vent, without any luck. Please Help!
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Old 11-25-2010, 09:08 PM
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Default Re: ProofReading Enzymes help!

Stratagene's PFU, right? Well, this Taq has a "special" protocol for it to work. I recommend you to look to the data sheet that comes with the enzyme. The protocol is specified there. Stratagene's PFU-HS II works at 68 C.

Anyway if you're not using this PFU, the protocol for your polymerase should come with the enzyme, or you can get it online.

Cheers!
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Old 12-27-2010, 04:53 PM
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Default Re: ProofReading Enzymes help!

This should probably be stickied somewhere in the forum as this kind of question has come up several times.

Pfu is less efficient and sensitive than Taq. Start with a range of temperature lower than the one you obtained with Taq, and don't skimp on the Pfu amount (follow the manufacturer's recommendations).
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Old 08-20-2012, 10:45 AM
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