User:Brian P. Josey/Notebook/2009/11/19

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Doing the Gel Again

We set up a gel today to double check to see that the PCR from Tuesday had failed. As you can see below, it did and the reaction has to be redone. In the picture only two out of the nine samples have an result. These are the samples with 4.5 mM and 5.0 mM concentration of Mg2+. In the picture the lanes, going from left to right, are:

  1. 1.0 mM Mg2+
  2. 1.5 mM Mg2+
  3. 2.0 mM Mg2+
  4. 2.5 mM Mg2+
  5. 3.0 mM Mg2+
  6. 3.5 mM Mg2+
  7. 4.0 mM Mg2+
  8. 4.5 mM Mg2+
  9. 5.0 mM Mg2+

Capture 00035.jpg
Anthony Salvagno 16:21, 19 November 2009 (EST):I had to do some heavy editing to make sure the image would be visible. The camera originally took a pretty dim picture.

Noticing that there is a small portion of the lane that has a sharp band, near the number two, and that there is a general smear, Ant suggested that the primers are attaching to both the specific point that we want, and nonspecific to other points. The Taq then binds to wherever the primer anneals and polymerizes from there. This results in the smear illustrated above. We believe this is from the temperature in the PCR machine being off during the annealing stage.

Redoing the PCR

Because of the failure of the last PCR, I am redoing it today, but lowering the temperature during the annealing stage to account for the errors that we are having with the temperature. The procedure is identical to the one that I did on Tuesday with the exception of the annealing stage. This stage is carried out at 50°C.

The exact volumes and concentrations of each of the reagents are:

{{#widget:Google Spreadsheet

key=tTLhOdy28Sc0gSWXsSgemXQ width=800 height=250


The programmed run times and temperatures are:

{{#widget:Google Spreadsheet

key=t-zsv6Zd6QUAs9pjnLKCpdg width=500 height=250


I won't know until I run a gel, probably tomorrow, if this reaction was successful. It should be noted however, that we made an error while programing the PCR and specified that the 94°C stage should proceed for thirty minutes and not thirty seconds. We caught this after three cycles and fixed it. So if the reaction fails tomorrow, then we know exactly why and I'll redo it as soon as I get a chance. I am starting to believe that the machine is jinxed.

Speaking of which:

Temperature Calibration

Both Anthony and I have taken time to go through the PCR cycle to check the temperature at each of the stages. While I've posted the raw data before, I have analyzed it paying special attention to the values for the annealing stage, when the plate needs to be at 60°C.

{{#widget:Google Spreadsheet

key=tPf2g1SVPbziXUFeX98vYdA width=800 height=500


Here is what is going on:

  • Average Annealing or Temperature at 60°C this is the raw average value that either Anthony or I measured.
  • Extra Points if you need it, for the samples where I measured multiple values for the temperature, I included all of them.
  • Average from 60 or Difference from 60 this is the difference between the measured value, and the desired value 60°C.
  • Average Omitting in an effort to remove error from the thermometer, we threw out some of the most extreme results, like 84°C, and any results with multiple determined values.
  • Average from 60 Omitting the difference between the omitted values and 60°C.

All the values across the top of the the table where detemined by me. The values in the lower left quarter where done by Anthony. The values in the lower right quarter are the averages of both Anthony's and my own data.