LAB 2 WRITE-UP
Thermal Cycler Engineering
Our re-design is based upon the Open PCR system originally designed by Josh Perfetto and Tito Jankowski.
The first piece of this PCR machine that we changed was the power supply source. We decided to make it smaller and more compact. A smaller power source 1), makes the entire machine lighter and adds mobility and 2), greatly reduces the chance of the power source overheating and causing the machine to melt or catch fire. As a result of making the power source smaller, we had to change the design of one of the side panels. The original side panels were crafted to fit the large power source. With the smaller power source, we had to re-align the holes so that they fit perfectly with our smaller power source. The biggest change we made to the PCR machine is changing the material from wood to ABS material. This material is similar to PVC material, but it is a cheaper substance to come by. A big advantage of a machine made of ABS material is that ABS is compatible with a 3D printer. That means that we can actually make PCR machines right in the lab because we have 3D printers.
The construction of the modified open PCR machine isn't any different than the original steps to putting it together, there are just modified parts like a smaller more compact power supply that is more efficient and the body is made out of ABS plastic which can be rapidly prototyped using 3-D printers so that they technology of open PCR can become more readily available. Here is a link to a guide to putting together your open PCR machine.
| Supplied in the Kit || Amount
| PCR Machine || 1
| Fluorimeter || 1
| Black Box || 1
| Positive Control DNA Sample|| 100 μL
| Negative Control DNA Sample || 100 μL
| 10 μM Forward Primer || 10 μL
| 10 μM Reverse Primer || 10 μL
| GoTaq Colorless Master Mix || 400 μL
| Eppendorf Tubes || 8
| Test Tubes || 8
| Pipettes || 10
| Hydrophobic Glass Slides || 5
| Tris Buffer (0.025% SYBR Green) || 100 μL
| Supplied by User || Amount
| DNA Samples || 200 μL
| Open PCR Software || 1
1. You will need a PCR machine and computer.
2. Download the PCR software onto the computer.
3.Thaw the GoTaq Colorless Master Mix at room temperature. Vortex the Master Mix, then spin it briefly in a microcentrifuge to collect the material at the bottom of the tube.
. Prepare the following reaction mix on ice:
| Reagent || Volume
| Template DNA (20 ng) || 0.2 µL
| 10 µM forward primer || 1.0 µL
| 10 µM reverse primer || 1.0 µL
| GoTaq master mix || 50.0 µL
| dH2O || 47.8 µL
| Total Volume || 100.0 µL
5. If using a cycler without a heated lid, overlay the reaction mix with 1-2 drops of mineral oil to prevent evaporation during thermal cycling. Centrifuge the reaction mix in a microcentrifuge for 5 seconds.
6. Place the reactions in a thermal cycler that has been preheated to 95 degrees Celsius. Perform PCR.
What Occurs In the PCR Machine
1. Denaturation: a 2-minute denaturation at 95 degrees celsius.
2. Annealing: perform the reaction about 5 degrees Celsius below the calculated melting temperature of the primers and increasing the temperature in increments of 1°C to the annealing temperature; this should occur anywhere between 30 seconds and 1 minute.
3. Extension: performed between 72-74 degrees Celsius, extension allows 1 minute for every 1 kb of DNA to be amplified; the suggested time for extension is 5 minutes.
4. Refrigeration: refrigerate the tubes at 4 degrees Celsius for several hours; this will minimize the opportunity for DNA polymerase to continue to be active at higher temperatures.
5. Cycle Number: the optimal amplification is 25-30 cycles, but up to 40 may be performed.
DNA Measurement Protocol
1. Turn on the excitation light using the switch for the Blue LED.
2. Place your smart phone on the cradle at a right angle from the slide.
3. Turn on the camera setting on the smartphone. Turn off the flash and set the ISO to 800 or higher and increase the exposure to maximum. You should also turn off the autofocus, if possible, and make sure that you can take an image where the drop on the slide will be in focus.
4. Adjust the distance between the smartphone on its cradle and the first two rows of the slide so that it is as close as you can get without having a blurry image.
5. The pipette should be filled with liquid only to the bottom of the black line. Then, carefully place two drops of water in the middle of the first two rows of the slide using the plastic pipette. Then add two more drops. The drop should then be pinned and look like a beach ball. It should be between 130-160 μL.
6. Align the drop by moving the slide so that the blue LED light is focused by the drop to the middle of the black fiber optic fitting on the other side of the drop (you will see that it has a small opening that is used for spectral measurement).
7. Cover the fluorimeter with the light box, but make sure you can access your smartphone to take the image. The light box should be used to remove as much stray light as possible, but do not worry if you have some light.
8. Take three images of the drop of water. Do not move your smartphone.
9. Remove the box and be careful not to move your smartphone. If you want to adjust for any movement, use the ruler provided to measure the distance so that you can return to that location. You can also use ImageJ to compensate for moving the camera, but it makes the analysis more complicated.
10. Use a clean plastic pipette to remove the water drop from the surface.
11. Push the slide in so that you are now in the next set of two holes.
12. Repeat steps 5-10 four more times so that you have now imaged all 5 positions on the slide.
13. Record the type of smartphone you used, the distance from the base of smartphone cradle to measurement device, and attach one image for each position of the drop.
Image J Procedure
1. Search Image J in Google and then download Image J
2. Open Image J then click "file" and click "open" and open the image you want to analyze
3. Once your image is open click "analyze" and then click "set measurements" and check the boxes "area" "integrated density" and "mean gray value" leave the rest of the boxes empty
4. now click "image" then click "color" and then click "split channels"
5. This will split your image into three, you will use the one that is marked as the "green" picture, cancel the others
6. Activate the oval tool
7. draw the best oval you can around the drop and then press the control button+ the "m" key
8. Move the oval over to the background (the black around the picture) and press the control button and the m key again
9. repeat steps for all pictures
10. Save your data in an excel format by clicking "file" and then clicking "save as" then save the file with the name you want
Research and Development
Background on Disease Markers
"GoTaqÂ® Colorless Master Mix (M714) Product Information." GoTaqÂ® Colorless Master Mix Protocol. Promega, 2012. Web. 15 Nov. 2012. <http://www.promega.com/resources/protocols/product-information-sheets/g/gotaq-colorless-master-mix-m714-protocol/>.
Hunt, Margaret. "Real Time PCR Tutorial." Real Time PCR Tutorial. University of South Carolina, 10 July 2010. Web. 15 Nov. 2012. <http://pathmicro.med.sc.edu/pcr/realtime-home.htm>.