Transferring liquids using the micropipette was fairly easy. When transferring the PCR reactions to the buffer, the value was set to 120 micro litters to ensure that all 100 micrometers of the PCR reaction is drawn up, because of this no liquid was left in the tube. The pre lab was helpful in that it advised to set the micropipette to a higher value so that no liquid is left in the tube. The two stops have two different purposes. The first one is to draw the designated amount of liquid, and then the second stop fully excretes the sample, leaving no liquid in the micropipette tip. The final reactions did have the same amount of liquid, and the labeling scheme remained the same, group name and patient sample or positive or negative control was labeled onto the tubes
Fluorimeter Procedure
Imaging set-up
The phone camera setting was first changed. iPhone cameras did not have the settings to change into the ones that the lab report required, so an android was used. The android phone, Motorola Z2 force, was changed to the settings recommended by the lab: ISO 800, White Balance on auto, highest exposure and highest saturation setting, and the lowest contrast setting. 160 micro liters of water was placed on the slide on the fluorimeter. The phone was placed on the stand and adjusted the distance between the slide and the phone until it was the image of the drop of water was focused. At around 10 centimeters distance from the phone and the slide, the camera focused.
Placing Samples onto the Fluorimeter
1. Attain a slide from the instructor. Detect the rough hydrophobic side and place it in the fluorimeter with that side facing up
2. Gather the samples of the different concentrations (5, 2, 1, .5, 2.5, 0) of calf thymus and the patient DNA samples including the positive and negative controls.
3. On the slide, there is a row of middle black dots, in between these dots is where the sample is to be placed
4.Micropipette 80 uL SBYR GREEN 1 in between the two black dots, then doing one sample at a time, micropipette 80 uL of the sample and discharge it on top of the drop of SBYR GREEN 1
5. Then, align this drop to the blue LED light that is located on the side.
6. Place the black box over the fluorimeter and the phone and take three images of the sample.
7. Label image accordingly
8. Set the micropipette to 160 uL and transfer the liquid from the slide into a designated waste bin.
9. The next sample can be placed in between next row of black dots.
10. Repeat these steps for all of the different calf thymus concentrations and the patient DNA samples and positive and negative control. Make sure that in each "trial" the phone is directly the same distance it was calibrated to focus in.
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
5
0.5
0
Calibrator Mean Values
Calibration curves
Images of Our PCR Negative and Positive Controls
Positive control
Negative control
PCR Results: PCR concentrations solved
PCR Results: Summary
Our positive control PCR result was 3208726.333 μg/mL
Our negative control PCR result was 6304994.667 μg/mL
Observed results
Patient 98625 : had a significant amount of SBYR GREEN 1 show up, a green dot in the middle.
Patient 72973 : had no SBYR GREEN 1 show up, just blue, and clear.
Conclusions
Patient 98625: Positive. from the image of the positive control this patient has the same prominent green dot.
Patient 72973 : Negative. from the image, this patient did not have a green dot and was just clear.
BME 100 Fall 2017
