User:Anthony Salvagno/Notebook/Research/2009/10/29/Thermal Cycler Thermocoupled

From OpenWetWare

Jump to: navigation, search

I don't know what the best way to approach this would be, but I will just take lots of measurements at different temperatures in different tube positions and see what the deal is. I will put all my readings in my patented spreadsheet.

Method

There are 20 positions a tube could be in in our thermal cycler. There are also a range of temperatures that can be used in various situations. Using a thermocouple I did a calibration measurement of the block in every tube position at 37C. By this I mean I put the temp sensor in a tube holder (on the block) and touched it at the bottom of block. I had it set at 37C for about 5 min before I measured everything. These results are first below and are denoted in the pink boxes.

Next I filled a bunch of 0.5ml tubes with water (about half way). Andy punched holes in the top of the tubes large enough for the sensor to fit inside. I then placed these tubes randomly about the block. See the drawing in the results section. The thermal cycler comes equipped with a sensor and we filled it with oil and placed it in tube position 2. I then calibrated the tubes of water at 37C by placing the sensor in the tube and pushed it as far down as it would go (to just below where 100ul would be). One tube of water was filled to 100ul and this was used to simulate PCR conditions. Initially this tube was placed in position 12.

I set the thermal cycler to program pRL574 and performed the PCR settings. I chose this because I wanted to reach a variety of temperatures and change them frequently. Also because my PCR hasn't been working and I want to know if this machine is reaching the desired temperatures.

I took 3 sets of measurements:

  1. PCR cycle was going, hot lid on, lid up. Each cycle I would take recordings from 3 different tubes, and every cycle I would change the test subjects. For these readings I would pick random subjects. I measured different water tubes, tube 12, and empty positions (on the block).
  2. PCR going, hot lid on, lid up. I tested tube 12 in various tube positions with the lid up. I would record tube 12 in a single position for one cycle taking measurements at each temp of the cycle.
  3. PCR going, hot lid on, lid down. I tested tube 12 in various tube positons with the lid down. I would record data for the tube temps in a single position for one cycle taking data at each temp of the cycle.

Typically I would record a temperature point when the time left at a given temp was within 10 sec of switching. For instance if the cycle was at 94C I would record a temp at t < 10s. At t = 0s the temp would switch to the next setting in this case 60C. For each set of measurements (see above) I restarted the PCR program.

Data is shown below.

Results

View/Edit Spreadsheet

Analysis

As of now I don't know what to do with the above information. Steve Koch 00:27, 30 October 2009 (EDT): These look like really important data, especially the last part where lid is closed. It's not clear to me how long the cycler had been at a particular setting before you recorded the measurement? This must have taken a ton of time, thanks for doing it! Steve Koch 11:51, 30 October 2009 (EDT): Unfortunately, I think only the "lid closed" are reliable. But looking at the last set, "lid closed / sim tube," sim tube is doing an awful job, especially at 94C. That is surprising. I made a 4th column in Excel that was the difference (measured temp - temp) and colored the negative numbers red, which made it easy to see the pattern. I think you should repeat this using "block" setting. But you don't have to do so many wells. I think you should do a couple wells with a water tube and a couple directly on the block.

Personal tools