The pre-lab reading was helpful in understanding exactly what needed to be done during the lab to assure to greatest accuracy while using the micropipette. The micropipette is pushed down to the first stop before inserting the tip into the reagents and releasing the stop to draw up the desired amount (which is pre-set by twisting the dial on the side of the micropipette before starting). After the desired volume of reagents have been drawn into the micropipette, the tip is then place into the properly labeled reaction tube and then the top is pushed down to the second stop to expel all liquid within the pipette tip. All of the final reactions should then have roughly the same volume of liquid. There was a minuscule amount of liquid left in the original tubes after using the micropipette to transfer the DNA samples or PCR reaction mix to the designated pre-labeled reaction tubes. It was unnecessary to alter the labeling scheme due to the fact that the reaction tubes were meticulously labeled in the exact same order that was determined in Lab Write-Up 4.
Fluorimeter Procedure
Imaging set-up
Get a smartphone with a camera and a smartphone (optional) for taking data. Record the model of the phones.
Inactivate the flash (necessary).
Set ISO to 800 or higher.
Set the white balance to auto.
Set the exposure to the highest setting.
Set the saturation to the highest setting.
Set the contrast to the lowest setting.
Steps 3-7 are optional. It is not necessary for your phone to be able to perform all of those actions.
Set a timer to take the picture after 3 seconds or more.
Placing Samples onto the Fluorimeter
Place a slide with the glass side on the bottom onto the fluorimeter.
Set the micropipette to 80 microliters and remember to dispose and use a new tip every time it is necessary.
Take 80 microliters of SYBR GREEN I and place it on the slide in between the first two rows.
Take 80 microliters of the 0 concentration 2X Calf Thymus DNA solution and place it into the SYBR GREEN I.
Turn on the blue light and line up the drop with the light.
Put the black box over the florimeter and take a picture and completely close the box. The camera should have a timer and finish taking the picture when the box is fully closed.
After taking three pictures, move the slide so that the light will line up between the next two rows. Use a new slide if you run out of room.
Remove anything on the slide and dispose of it.
Repeat steps 2-8 with different concentrations of 2X Calf Thymus DNA solution.
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
High Calf Thymus DNA: 5 μg/mL sample
Low Calf Thymus DNA: 0.5 μg/mL sample
Zero Calf Thymus DNA: 0 μg/mL sample
Calibrator Mean Values
Table 2: Calibration Data
Calibration curves
Images of Our PCR Negative and Positive Controls
Positive Control
Negative Control
PCR Results: PCR concentrations solved
Table 5: PCR Solved
PCR Results: Summary
Our positive control PCR result was 2.303 μg/mL
Our negative control PCR result was 0.669 μg/mL
Observed results
Patient 20268 : This patient tested positive for the disease we were testing for. Their SYBR Green I levels were far closer to that of the positive control than the negative control, showing a strong green hue in the tested droplet. This can be seen in the data, as the μg/mL of the positive control was found to be roughly 2.3, and in the case of Patient 20268, the average was found to be roughly 2.50 μg/mL.
Patient 49271 : This patient tested negative for the disease. Their SYBR Green I levels were about the same as the negative control - this was seen by the lack of a severe green hue in the sample. They tested similarly to the negative control, which had a μg/mL of roughly 0.669. The average in Patient 49271 was roughly 0.69 μg/mL
Conclusions
Patient 20268 : This patient tested positive for the disease, as compared to the positive control - the patient’s μg/mL was very similar, being only roughly 0.2 off from the positive control, and about 1.8 off from the negative control.
Patient 49271 : This patient tested negative for the disease, as compared to the negative control - the patient’s μg/mL was very similar, being only roughly 0.2 off from the negative control, and about 1.7 off from the positive control.
Extra Credit: Gel Electrophoresis
Gel Electrophoresis:
BME 100 Spring 2017
