In pipetting the samples to set up the reaction, the pre-lab reading was very helpful. In addition to this, some members of our group had previous experience in pipetting, which was also helpful in properly pipetting the samples. Distinguishing between the first and second stop on the pipettes was important. The first stop was used when picking up the liquid, to ensure that there wasn't an excess of liquid being picked up. The second stop on the pipette was used to deposit the liquid out, this was to make sure that all of the liquid was released from the pipette. The final reactions did not have the exact same amount of liquid because when pipetting sometimes the pipette would not completely pick up the full amount, or after releasing there would still be some liquid in the pipette. The labeling scheme made it very easy to determine which tubes were ours and which were not ours. Also, the labeling made it easy to determine which were the controls, and which belonged to which patient.
Fluorimeter Procedure
Imaging set-up
1. We inserted the iPhone 6 into the cradle, and had one person hold the base of the cradle for stability.
2. We placed a plastic box underneath the fluorimeter so that the iPhone was correctly lined up.
3. We measured the distance between the iPhone camera and the drop, the distance was 5 cm.
Placing Samples onto the Fluorimeter
Using a micropipette, place 160μL of water on the slide between the first two rows.
Adjust the location of the slide so that the water drop is directly between the light.
Measure the distance between the camera and the drop of water
Pick up the drop of water using the micropipette and discard into the waste cup.
"Using the micropipette, place 80μL of SYBR Green I on the slide between the first two rows."
"Place 80μL of the 2.5μg/L concentration calf thymus solution."
"Focus the camera on the drop to make sure that it is clear, turn on the 3 second timer on the phone, press the button, and close the lid of the box"
"Take two more pictures of the same drop."
"Open the lid of the box, and discard the 160 μL of solution into the waste cup."
"Repeat steps 5-9 for the remaining concentrations of calf thymus solution."
"Create solutions with the PCR reaction samples. To do this, place 100μL from the PCR tube into a tube of 500μL of buffer. Invert the tube to ensure that the solution is mixed. Create solutions for both of the controls and the patient data."
"Repeat steps 5-9 for the PRC/buffer solutions."
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
High Concentration
Low Concentration
Zero DNA
Calibrator Mean Values
Calibration curves
Images of Our PCR Negative and Positive Controls
Positive
Negative
PCR Results: PCR concentrations solved
PCR Results: Summary
Our positive control PCR result was 4.401 μg/mL
Our negative control PCR result was 1.227 μg/mL
Observed results
Patient 18158 : 3.215μg/mL. The images appeared to be green and glowing.
Patient 76712 : 1.527μg/mL. The images appeared to be dark and not glowing.
Conclusions
Patient 18158 : Positive. The value of concentration is closest to that of the positive control. The images also appeared to be most similar to the positive control, therefore it can be assumed that Patient 18158 is positive.
Patient 76712 : Negative.The value of concentration is closest to that of the negative control. The images also appeared to be most similar to the negative control, therefore it can be assumed that Patient 76712 is negative.
BME 100 Fall 2016
