Pipetting the samples was a very easy process. The pre lab reading and video helped a great deal. The video made it very easy to learn how to properly pipette. The differences between the first and second stop on the pipette is that they serve different purposes. The first stop allows you to "suck up" the exact quantity of liquid that is specified on the pipette. The second stop is for pouring the content of the pipette into a container. The second stop ensures that all the contents in the pipette will be removed. Some final reaction mixtures seemed to have a very slight difference in the amount of liquid, however there was no liquid left in the tubes that had the DNA samples and PCR reaction mix. Our group did not have to change the labeling scheme at all. The one provided proved sufficient.
Fluorimeter Procedure
Web camera set-up
The camera used in this lab was from a HTC Desire 626 smartphone. Prior to placing the phone in the cradle we adjusted a few settings on the camera. We inactivated the flash, set ISO to 800, set white balance to auto, set exposure and saturation to the highest settings, set contrast to the lowest setting and finally we set the timer to 5 seconds. The phone was placed in the cradle so that it was perpendicular to the fluorimeter. Plastic trays were used to raise the fluorimeter in order to take a "sideways" picture of the drop. The camera was placed as close to the fluorimeter as possible without the image coming out of focus, this distance was approximately 7 cm.
Placing Samples onto the Fluorimeter
Place a slide onto the Fluorimeter so that the smooth side is facing downwards and the rough side is facing upwards.
Using the pipettor, place an 80 microliter drop of SYBR GREEN in between the first two rows of the slide. The drop should bead upwards, making it look like a ball.
Place an 80 microliter drop of the solution to be tested onto the drop of SYBR GREEN. This is now the sample drop.
Adjust the slide so that the sample drop is aligned with the Blue LED light.
After completion of the pictures, use the micropipette to remove the 160 microliters of liquid from the slide and move the slide into the next position.
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
High:
Low:
Zero:
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 7.453 μg/mL
Our negative control PCR result was -3.198 μg/mL
Observed results
Patient 51714 : Pictures for patient 51714 tended to show more green and appeared brighter when the Blue LED was applied. The initial PCR concentration values were also larger.
Patient 78326 : Pictures for patient 78326 tended to show more blue and less green when the Blue LED was applied. The initial PCR concentration values were also smaller.
Conclusions
Patient 51714 : Positive. Although two samples tested negative, the third was positive. The two negative samples also had closer values to the positive control concentration than those of patient 78326 did. The images for this patient also appeared more green in color, similar to the positive control.
Patient 78326 : Negative. The samples all tested closer to the negative control concentrations than the positive control concentrations. The images also appeared more blue in color, similar to those of the negative control.
BME 100 Spring 2016
