Our teams experience with pipetting the samples to set up the reaction was very easily understood. The pre-lab reading really helped understand the basic directions on what to do during the lab, but mainly the demonstration in class was the most helpful. We understood the difference between the first and second stop on the pipettor and that was very easily managed. The final reactions did have exactly the same amount of liquid of 160 micro-liters. If there was any liquid left in the pipette tubes it was a very small and insignificant amount of maybe a small droplet. We did have solution left in the tubes that had our DNA samples, but we did run completely out of SYBR Green I solution by the end. We did not change our labeling scheme from the previous lab.
Fluorimeter Procedure
Imaging set-up
Set up a smart phone to take pictures of the droplets. Before taking the picture, inactivate the flash of the camera and set the timer to 3 seconds or more.
After setting up the drop on the fluorimeter, set up the phone so that it will take a picture of the droplet from the side. Bring the phone to the drop as close as possible to a point before the image turns blurry (the phone will be at least 4 cm away), and record the distance away for each trial.
Make sure the blue LED light is lined up to directly shine the drop and then place a box to cover the fluorimeter and the camera, while trying to keep any light from coming in.
Take 3 separate images while making sure that the neither the drop's or the phone's disposition has been disturbed.
Remove the box, remove the drop, and then repeat the procedure with the other liquids.
Placing Samples onto the Fluorimeter
Step one, with a clean micropipette tip, use the pipettor to obtain 80 micro liters of the H2O sample.
Step two, place the sample onto the slide, placing the droplet between two dots within the first row.
Step three, eject the used pipettor tip into the red solo cup.
Step four, with a clean micropipette tip, obtain 80 micro liters of the SYBR GREEN 1 solution.
Step five, place the tip of the pipettor into the existing sample droplet.
Step six, release the SYBR GREEN 1 solution into the droplet.
Step seven, eject the used pipettor tip into the red solo cup.
Step eight, after using the smartphone to capture an image of the droplet
Step nine, with a clean micropipette tip, retract the droplet into the pipetter and eject into the red solo cup.
Step eleven, repeat this action until all the existing sample is eliminated from the slide.
Step twelve, repeat steps 1-11 with the remaining samples, moving the droplet to different row positions per sample.
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
5 μg/mL sample
0.5 μg/mL sample
zero DNA
Calibrator Mean Values
Calibration curves
Images of Our PCR Negative and Positive Controls
Negative control PCR sample
Positive control PCR sample
PCR Results: PCR concentrations solved
PCR Results: Summary
Our positive control PCR result was _.7___ μg/mL
Our negative control PCR result was _-2.3___ μg/mL
Observed results
Patient _21428____ : This image contained no significant coloring from the SYBR GREEN 1 solution, having a PCR product concentration of -1.5 mL.
Patient _85431____ : This image contained no significant coloring from the SYBR GREEN 1 solution, having a PCR product concentration of -2.1 mL.
Conclusions
Patient _21428____ : Compared to the positive control value of .7 mL, and the negative control value of -2.3 mL, patient 21428's value of -1.5 mL confirms there is no evidence of the targeted DNA.
Patient _85431____ : Compared to the positive control value of .7 mL, and the negative control value of -2.3 mL, patient 85431's value of -2.1 mL confirms there is no evidence of the targeted DNA.
BME 100 Fall 2017
