LAB 5 WRITE-UP
PCR Reaction Report
Our group had a successful experience in pipetting the samples to set up the PCR reaction. The pre-lab reading was helpful because it taught how to use a pipettor and the common mistakes to avoid. In lab it was easy to understand the difference between the first and second stop on the pipettor because it was easy to feel when the first stop had been reached, and then to press on to the second stop. The final reactions did not have the same amount of liquid because there were small amounts of DNA sample and PCR reaction mix left over in all of the tubes. While these leftover amounts were small, they did vary slightly from tube to tube. Finally, our group was able to keep our original labeling scheme because it worked effectively with our data and samples.
Smart Phone Camera Settings
- Type of Smartphone: iPhone 5
- Flash: Off
- ISO setting: 800
- White Balance: Auto
- Exposure: Highest Setting
- Saturation: Highest Setting
- Contrast: Lowest Setting
- To set up the camera, place the slide and the slide holder in the Fluorometer, and then place the smart phone cradle into the box. Once both of these items are in the fluorometer, place items under the slide holder to raise it so that the slide is at a 90 degree angle to the smart phone camera. Then pipette a 160μL drop of water onto the slide. Set up the camera settings and place the phone into the phone cradle. Adjust the cradle and phone until the image on the phone is clear, and is no closer than 4cm to the slide holder. Once this is done, remove the phone, but keep the cradle in its place.
- Distance between the smart phone cradle and drop = 5.5cm
Placing Samples onto the Fluorimeter
- Step 1: Test out the setting and placement of the camera by using a 160 μL drop of water on the slide. The drop should be in the middle of the first two rows of the slide.
- Step 2: Adjust the placement/ distance of the camera from the slide so that the camera is as close as it can be with the image still coming out clear. Record.
- Step 3: Once the phone cradle is set up in the right place, use the micropipettor take an 80 μL sample of SYBR GREEN solution and place it on a slide. Then take an 80μL sample of .25 Conentration of 2x Calf Thymus DNA solution and place it on top of the SYBR GREEN solution.
- Step 4: Place smart phone back in place.
- Step 5: Close the lid of the fluorometer and take three pictures of the solution.
- Step 6: Repeat steps 3-5 for the Calf Thymus DNA concentrations of 0.5, 1, 2, 5.
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
- Order of images left to right: 0.5 μg/mL, 5 μg/mL, and zero DNA
Calibrator Mean Values
Images of Our PCR Negative and Positive Controls
- Order of images left to right: positive control, negative control
PCR Results: PCR concentrations solved
PCR Results: Summary
- Our positive control PCR result was μg/mL
- Our negative control PCR result was μg/mL
- Patient 21956 : The image showed a green oval inside of the drop. The average is 80.976.
- Patient 84919 : The image showed no green oval inside of the drop. The average is 69.6214.
- Patient 21956 :Patient has higher PCR concentration so the DNA displays the sought after mutation for disease.
- Patient 84919 :Patient has lower PCR concentration so the DNA does not displays the sought after mutation for disease.