Pipetting was very easy overall. The virtual labs helped to walk through each step in order to allow practice before the real application. At first, the second stop was a little confusing because it stops at the first stop and almost seems like all of the solution had been released and there was hesitation on when to push the button or when to release. Also, we were unaware of hoe to dispose of the tip from the micropipette by the button but figured it all out eventually. The final reactions did not have the same amount in the PCR tubes and some of the mix and samples had remaining liquid which was concerning. The labeling was very consistent as easy to monitor.
Fluorimeter Procedure
Imaging set-up
The fluorimeter was rested on top of a stack of plates so the camera and the fluorimeter glass were about equal heights. The stack of plates were between 35 and 45. The slide was inserted into the fluorimeter with the smooth side down and at a position where the blue light hit between the first two drops. The iPhone 6 was placed inside the phone cradle approximately 4cm away from the location of the drop. The drop would be placed in the middle column and between the first two rows of the glass slide. The light box was placed around the fluorimeter with one flap up to allow the photo to be taken. The camera was set on a 10 second timer so there was time to close the flap without disrupting the camera's focus.
Placing Samples onto the Fluorimeter
Set the micropipette to 80microliter
Add a new pipette tip to the micropipette
Press the micropipette to the first stop, insert tip into SYBR GREEN I Dye, fully release button, and remove pipette from the solution
Press the micropipette to the second stop in the middle of the first two rows of the slide, pull away pipette and release button, and eject tip into the waste
Add a new pipette tip to the micropipette"
Press the micropipette to the first stop, insert tip into calf thymus/water blank/PCR solution, fully release button, and remove pipette from the solution
Press the micropipette to the second stop in the middle of the first two rows of the slide adding onto the already present drop, pull away pipette and release button, and eject tip into the waste
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
5μg/mL
0.5μg/mL
0μg/mL
Calibrator Mean Values
Calibration curves
Images of Our PCR Negative and Positive Controls
+
-
PCR Results: PCR concentrations solved
PCR Results: Summary
Our positive control PCR result was 13.53578461μg/mL
Our negative control PCR result was 2.110732165μg/mL
Observed results
Patient 63747: The drop appeared to be very green. In the split channel image, the drop appeared very bright. The initial concentration was in the 50-60μg/mL range.
Patient 53529: The drop appeared to be almost transparent. In the split channel, the drop appeared more dark. The initial concentrations came out to be 3-9μg/mL.
Conclusions
Patient 63747: The concentration was well over the positive control which shows that the patient tested positive for the DNA because the sample showed large amounts of DNA produced.
Patient 53529: The concentrations were between the positive and negative concentration but never reached the positive threshold. The patient is to be negative because they did not produce large amounts of thee DNA.
BME 100 Fall 2016
