5 replies to this topic

[thegradstudent]

Hi !

I'm looking for high fidelity RT PCR enzymes with proofreading capacity. What is your enzyme of choice?

Thanks in advance

[ana]

thegradstudent, on Feb 23 2005, 04:19 PM, said:

Hi !

I'm looking for high fidelity RT PCR enzymes with proofreading capacity. What is your enzyme of choice?

Thanks in advance

I am working with Sensiscript RT Kit  and ProofStart Polymerase in two-steps RT-PCR and I´m getting good results with them. Both are fron Qiagen. In Qiagen web you can find its characteristics.
I hope it helps.

[seqgirl]

Superscript III One-Step RT-PCR System with High Fidelity Platinum Taq from Invitrogen rocks.

Edited by seqgirl, 24 February 2005 - 06:06 AM.

[jadefalcon]

I had good results with the Titan-One-Tube HiFi RT-PCRT kit from Roche

mike

[kga1978]

Hmmm as far as I know there is no such thing as a "proofreading" RNA dependent DNA polymerase. At least that would not be of any advantage to the retro-virus producing it. However some companies may have made blends that will work with proofreading activity?

Anyways - my suggestion is to use any MLV based RNase H negative RT enzyme - e.g. SuperScript from Invitrogen.

If you need to do a downstream PCR for cloning then use any of the High Fidelity blends around. My suggestion is a new Novagen blend called "KOD Polymerase", which is the most amazing blend I have seen around. First of all the fidelity is amazing, second of all the processivity is just out of this world - 10kb can be done with an elongation step of less than 1 min!!! Also this blend contains antibodies to the two enzymes, so the mix will work as a "HotStart".


Good luck!  

[jadefalcon]

I think the proofreading refers to the DNA-dependent DNA-polymerase part of the RT-PCR kits, not to the reverse transcriptase.....

mike