Aug 26, 2015
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Medical Student (Accepted)
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?
 
Dec 8, 2014
385
164
Status
Pre-Medical
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.
 
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echo87

5+ Year Member
2+ Year Member
Jun 15, 2014
252
172
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Medical Student (Accepted)
For the future: Google, ResearchGate, PubMed

In that order
 

Goro

7+ Year Member
Jun 10, 2010
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Somewhere west of St. Louis
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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?