PCR denaturing temp

[Fuculokinase]

I'm applying to an MS/DO program and I wanted to help out in a laboratory beforehand to get a good feel of a particular lab I'll be working in, but I'm actually now conducting research under a professor. I don't mind it, especially since I could get published, but it's been two years since I graduated and I didn't realize how much I forgot. For instance, I can't for the life of me remember how many micrograms of what I'm supposed to add to a PCR reaction. The professor showed me exactly what she wanted and at what degrees/minutes, and one of them throw me off. In a particular PCR reaction I set up last week the denaturing step is at 98°C compared to what I was taught which was 94°C. My question is strange and may be totally pointless for a premedicine forum since it's mainly just a question to appease my curiosity at 1am, but I was wondering if temperatures were set up differently for specific polymerases. I'm using pyrococcus furiosis compared to thermus aquaticus. Do they work best at different temperatures?

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[becomingdoctor]

Denaturing temp is usually 92-95 depends on GC ratio. Polymerase usually don't affect denature temp, rather, manufacture will give you recommended elongation temp, usually 68-72. If you are troubleshooting your pcr, always start with gradient pcr. It will be much helpful if you post it on research gate.

 

[echo87]

For the future: Google, ResearchGate, PubMed

In that order

 

[Goro]

I use 94 or 95 degrees for all for my denaturing steps.

I'm applying to an MS/DO program and I wanted to help out in a laboratory beforehand to get a good feel of a particular lab I'll be working in, but I'm actually now conducting research under a professor. I don't mind it, especially since I could get published, but it's been two years since I graduated and I didn't realize how much I forgot. For instance, I can't for the life of me remember how many micrograms of what I'm supposed to add to a PCR reaction. The professor showed me exactly what she wanted and at what degrees/minutes, and one of them throw me off. In a particular PCR reaction I set up last week the denaturing step is at 98°C compared to what I was taught which was 94°C. My question is strange and may be totally pointless for a premedicine forum since it's mainly just a question to appease my curiosity at 1am, but I was wondering if temperatures were set up differently for specific polymerases. I'm using pyrococcus furiosis compared to thermus aquaticus. Do they work best at different temperatures?

 

[Dr. Death]

This could also be elevation dependent if you are at >4000 feet