12 December 2016 5 9K Report

Good afternoon,

I have two main questions. I am working on the amplification of several mitochondrial and ribosomal markers for fish DNA. As I want to sequence afterwards, I was looking for a PCR enzyme with a high fidelity. I saw the Platinium SuperFi (Invitrogen) and got a free sample to test my primers (3 mitochondrial, 1 ribosomal). I tested these 4 markers and only one worked (clear band on the gel after electrophoresis).

These primers are coming from the literature and have been used on fish previously so I am wondering if the problem comes from the PCR conditions. Indeed, Tm given by the calculator specific of this enzyme are much higher (sometimes 10° difference) than the Tm found in other papers. I called the technical support related to this and they advised me to only test the Tm given by the calculator and eventually 2 degrees less. One of my colleagues just advised me to go for 5°C less and that it might be the reason why I didn't see anything for 3 markers (Tm too high). Has anyone got experience with this enzyme and knows if that could be the problem ?

Second question, it seems that this enzyme is a bit too much for what I want to do after with the PCR products. I am currently looking for PCR enzymes less specific, but still good enough to have good results (error rate low enough).  I saw the HotStar HiFidelity Polymerase Kit (QIAGEN) and the Phusion Hot Start II High fidelity (ThermoFisher). Has anyone any other suggestions ? (PCR product sizes : between 500 and 800 bp).

I would be very grateful if someone can help me or give me an advice.

Thanks !

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