User:Anthony Salvagno/Notebook/Research/2011/02/03/Thermal Cycler Analysis and pBSTXI PCR Try 3

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Thermal Cycler Analysis

I have known for some time that the thermal cycler wasn't very accurate. I tried to determine how off it was before but that was a mess. This time I had help.

I used the temperature controller we bought back in June of last year to read and record the temperatures it measured. I ran the thermal cycler a total of 4 times. The first time I ran the program as is with my probe in the same reaction tube I usually work with. For trial 2 I bought thin-walled reaction tubes and used that with everything else the same as in trial 1. Trial 3 I changed the PCR program to use the temperature reading from it's own tube instead of simulating the tube temperature. For trial 4 I changed the temperature settings based on observations from trials 1, 2, and 3 and from messing around with the machine myself.

Results

Trial 1:

  • PCR Settings -- melt at 94C, anneal at 60C, extend at 72C; temp readings: Sim Tube
  • Actual recorded temps:
    • 94C -> ~92C avg
    • 60C -> ~70C avg
    • 72C -> ~77C avg
  • The thermal cycler would get up to temperature and start the cycle clock before the thermister registered anywhere near the cycle temp. This would cause the temperature time to be much less then expected. Also the temps were way off and not optimal for PCR reaction.

Trial 2:

  • PCR Settings -- melt at 94C, anneal at 60C, extend at 72C; temp readings: Sim Tube; used thin-walled reaction tube
  • Actual recorded temps:
    • 94C -> 92C avg
    • 60C -> 65C avg
    • 72C ->75.5C avg
  • The times at each temp were less as per above but they weren't as short as above. Also the tube would reach temperature faster with less exponential curve. Finally the temps were closer to the indicated temp but still not perfect.

Trial 3:

  • PCR Settings -- melt at 94C, anneal at 60C, extend at 72C; temp readings: Tube
  • Actual recorded temps:
    • 94C -> 92C avg
    • 60C -> 65C avg
    • 72C ->75.5C avg
  • Mostly the same as trial 2, but the tube would be at its final temperature for longer and much closer to the targeted time as the setting.

Trial 4:

  • PCR Settings -- melt at 95C, anneal at 50C, extend at 69C; temp readings: Tube
  • Actual recorded temps:
    • 95C -> 94C avg
    • 50C -> 59C avg
    • 69C ->73C avg
  • For this I guesstimated the temperatures that would give me the optimal conditions and I was luckily right. I changed the cycler program accordingly.

Raw Data

PCR Try 3

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