The dry materials were obtained from the front of the classroom. They consisted of a micropipetting device, a disposal beaker, a PCR tube rack, a strip of empty PCR tubes, and disposable pipette tips. Then, the wet materials were obtained. The wet materials consisted of the PCR reaction mix, the two different DNA primer mixes with their individual patient ID, and the positive and negative controls. Each of these samples contained 50 μL of solution and was held in a closed PCR tube. All of the PCR tubes that held the solutions were placed in the rack and identified. The strip of tubes originally had 8 connecting tubes, and we had to cut the strip in half to be able to fit in the thermal cycler. The empty tubes were labeled by Emily according to the solutions they were about to be holding. 50 μL of PCR solution was added to each empty tube using correct pipette techniques and with a new disposable tip each time. Emily did the first 4 tubes, and Calvin did the last 4. After each use, the pipette tip was disposed of in the disposal beaker. Following the addition of PCR mixture in each tube, the respective solutions were added. The tube labelled “G 10 P” received positive control solution, and the tube labelled “G 10 N” received negative control solution. The tubes labelled “G 10 1-1”, “G 10 1-2”, and “G 10 1-3” received the solution labelled with the first patient’s ID number (61026). The tubes labelled “G 10 2-1”, “G 10 2-2”, and “G 10 2-3” received the solution with the second patient’s ID number (86834). Once the solutions were correctly added two each tube, they were closed and added to the thermal cycler with Group 7’s tubes. The thermal cycler was programmed with the characteristics described in our first lab report with the help of Dr. Garcia. Upon closing the thermal cycler, our group cleaned up our work station and disposed of all the materials.
The experiment itself was completed by Emily and Calvin. This was Emily’s first time using micropipette techniques and thermal cyclers, but Calvin explained that he had previous experience with the devices. He was able to assist and guide Emily when she had trouble using the micropipette. Initially they had some problems collecting the solution without air bubbles. After a few tries, they were able to correctly gather the whole 50 μL. After closing the tubes, they made sure to check for consistency and that each tube held the correct volume of solution. Doctor Haynes and Doctor Garcia very helpful and gave very clear instruction.
Our complete PCR reaction tubes in a thermocycler.
Calvin and Emily posing with our PCR reaction.
Procedure
Camera Set-up
SMARTPHONE 1: iPhone X
Camera Settings
A. Inactivate the flash
B. Set ISO to 800 (or higher, if possible)
C. Set white balance to auto
D. Set exposure to highest setting
E. Set saturation to the highest setting
F. Set contrast to lowest setting
Wet-lab Procedure
1. Place a 160 microliter drop of water in the middle of the first two rows of the slide using the pipettor.
2. Turn on the excitation light using the switch for the Blue LED.
3. Turn on the camera on the smartphone and make sure the settings are identical to steps A-F above.
4. Place the smartphone on the cradle at a right angle from the slide. Adjust the height of the fluorimeter using the plastic trays so that the camera takes a picture of the drop sideways.
5. Adjust the distance between the smartphone on its cradle and the first two rows of the slide so that it at least 4 cm away from the drop.
6. Record the distance between the smartphone cradle and the drop using a ruler. Do not move the camera, cradle, or fluorimeter because the light collected will change slightly if there is a significant difference from one image to the next in the distances.
7. Place an 80 microliter drop of SYBR GREEN in the middle of the first two rows of the slide using the pipettor. Then, add 80 microliters of one of the calf thymus (or water blank) solutions listed above. This is now considered a "drop."
8. 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.
9. Use the timer on the smartphone camera so a picture can be taken after covering the fluorimeter and camera with the light box. The light box should be used to remove as much stray light as possible.
10. Take three images of the drop, make sure that the drop is focused.
11. Remove the box, being careful not to move the smartphone. If there is any need for movement, use the ruler to adjust for movement so that it can be returned to original position.
12. Use the pipettor to remove the 160 microliter drop from the surface and move the slide to the next position.
13. Repeat steps 7-12 for the other concentrations of calf thymus DNA.
Image J Procedure
1. Open Image J software.
2. Set Measurements: Click area, integrated density, mean grey value.
3. Open image and select image > color > split channel.
4. Using the green image draw and oval around drop.
5. Store in ROI Manager by selecting edit > selection > add to manager.
6. Repeat steps 3-5 for all images.
7. Analyze image by selecting analyze > measure.
8. Move all ovals to just above the drop.
9. Analyze background.
Data Analysis
Calibration
Calibration Curve 1
Calibration Curve 2
The first image, Calibration Curve 1, shows the graph of the Mean RAWINTDEN drop plotted against the Initial Calf Thymus DNA Concentration.
The second image, Calibration Curve 2, is the same graph without the highest initial concentration.
BME 100 Fall 2018
