User:Brian P. Josey/Notebook/2009/11/18

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Checking Yesterday's PCR

Anthony is showing me how to check the PCR from yesterday with a gel. We made a gel, and filled the wells up with 5 μL of the solutions from the PCR, and 1 μL of the 6x dye. Then we fill up the gel holder with TAE and apply a voltage across the gel. This then forces the gel to develop. We visualize it and take a picture to see if it worked.

Here is the picture from the gel. The ladder is on the left side, and it appears that only the 4.5 mM of Mg2+ developed, which is on the right side. We had hoped to develop the ladder and nine different samples, but only the one sample developed. Anthony thinks that it could be the result of us using so little DNA. Normally we would throw out all the bad samples, but here we decided to increase the amount of DNA in the gel and run another gel. We are going to do that tomorrow.

Ideas

There is the chance that the reaction just did not take place, with the exception of the 4.5 mM sample. This could be the result of the thermal cycler not heating and cooling the samples properly. So I need to look at my numbers from when we did the temperature check, and find the average values that the plate gets to for each of the set temperatures and the standard deviations each of them. It could be that the hot plate just doesn't cool down enough to do the annealing phase, so we might have to change the programmed temperatures to hopefully get the temperatures that we want. I also need to throw together another PCR reaction set up for tomorrow, just in case the one from yesterday turns out to not be successful.

Anthony Salvagno 13:53, 18 November 2009 (EST):I think you got it right. It is more than likely that the PCR just failed in every instance, but we know for a fact there is a temp that will get it to work at smaller molarities than 4.5mM. So here is the official write up of what we will do tomorrow:

  • Run a new gel with more DNA (15ul of DNA).
  • Set up same PCR as yesterday, but with different temperatures.
  • Tonight or before you come in tomorrow analyze your numbers for the annealing phase of PCR (thermocouple readings)
    • It would be great to see (1)average temp, (2)std dev from that temp, (3)closest tube pos to that temp (which tube holders get the closest to 60C)
  • From that we can figure out what temp to do for the PCR annealing. My gut tells me this is where the problem is.


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