For our pipetting the samples we used all of the liquid so we didn't have any problems with that. The pre-lab reading was very helpful. It explained how to draw the solution and place it back to the test tube. When actually touching the pipettor, we could see the distinct difference on it. The first stop was to take out the liquid and the second was to make sure you released all of the liquid. The final reactions mostly had the same amount of liquid. There was a little bit of liquid left in the DNA and PCR reaction mix. Yes, we did have to change our labeling scheme. We ended up switching patient 1 and patient 2 labels.
Fluorimeter Procedure
Smart Phone Camera Settings
Type of Smartphone: iPhone 6
Flash: N/A
ISO setting: N/A
White Balance: N/A
Exposure: N/A
Saturation: N/A
Contrast: N/A
Camera set-up
First, place to light box on the table or bench in front of you so that when turned on the light beam forms a horizontal line, parallel to the edge of the desk. Next, place the phone holder flush against the side of the light box that is facing you. Place a glass slide in the slot on the light box and adjust so the the edge is aligned with side of the box that is facing you. Then, pipet 180ul of distilled water on the middle hole of the first row and turn on the light. Adjust the glass slide so that the light beam goes through the center of the water drop. Place your phone upright in the holder with the camera facing the drop. Your phone should be flush against the side and not an angle. If needed, adjust the height of the phone so that it is parallel with the drop. This can be done by placing various item such as pipet tip boxes under the phone stand.
Distance between the smart phone cradle and drop = 8cm
Placing Samples onto the Fluorimeter
[Pipet 80 microliters of SYBR Green in the middle of the first two rows or the same place where you previously pipetted the drop of water.]
[Dispose of pipet tip in designated "tips" cup.]
[Pipet 80 microliters of distilled water into the same drop and dispose of tip in designated cup.]
[Adjust the glass slide so that the drop is aligned so that the light beam goes through the middle of the drop.]
[Using a camera phone, take three photos of the drop.]
[Using a new pipet tip, remove the liquid from the glass slide and transfer to the designated "liquid" waste disposal cup. Do this twice in order to remove all 180 microliters. Then dispose of tip in designated cup.]
[Replace the glass slide with a new one and repeat steps 1-6 using the following DNA calf thymus concentrations; .25ug/ml, .5ug/ml, 1ug/ml, 2ug/ml, 5ug/ml]
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
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 179.284μg/mL
Our negative control PCR result was 112.497 μg/mL
Observed results
Patient 53807 : The greater the concentration of PCR, the brighter the glow of green appeared. For patient one, we got an average concentration of 26.663ug/mL.
Patient 45421 : Similarly, the greater the concentration of PCR in the drop, the brighter the green glow appeared. We received an average concentration of 57.337ug/mL.
Conclusions
Patient 53807: Comparing to the positive and negative controls, patient one was negative.
Patient 45421: Patient two was also negative when compared to the controls.
BME 100 Fall 2015
