Name: Jose Luis Rivera
Role(s): Micropipetting, Fluorimeter Procedure, and Data Analysis
Name: Amity Jackson
Role(s): Data Collection and Analysis
Name: Jarrett Eshima
Role(s): Assisted with Micropipetting, Fluorimeter Procedure, and Data Analysis
Name: Sheldon Cummings
Role(s): Data Collection and Analysis
Name: Diba Pourazar
Role(s): Collecting materials and Data Collection and Analysis
Name: Katarina Junio
Role(s): Micropipetting, Fluorimeter Procedure, and Data Analysis, PCR Reaction Report
LAB 5 WRITE-UP
PCR Reaction Report
The pre-lab readings and activities were helpful in teaching us how to micropipette. It reiterated the steps in order to instill in our heads exactly how to micropipette, since PCR deals with very precise samples. We learned the difference between the first and second stop on the pipettor, in that the first stop is used for collecting the sample, while the second stop is used for releasing the sample.
In terms of actually collecting the samples, the final reactions had the same amount of liquid, with the exception of our positive control (G10+). That reaction had slightly less liquid, due to the positive control DNA sample having less than 50 μL in the tube. There was also a little bit of liquid left over in some of the PCR reaction mix, but it was only very little, being just a single drop at the very bottom of the tube. We also did not have to change our labeling scheme.
Smart Phone Camera Settings
- Type of Smartphone: Samsung Galaxy S5
- Flash: Off
- ISO setting: Auto
- White Balance: Auto
- Exposure: Max Value +2.0
- Saturation: N/A
- Contrast: N/A
- Place the phone in the phone cradle.
- Adjust camera setting as listed above.
- Adjust height so that the droplet is leveled with the camera.
- Measure the distance from the camera to the droplet and keep it constant.
- Distance between the smart phone cradle and drop = 9 cm
Placing Samples onto the Fluorimeter
- Place the slide in the Fluorimeter smooth side down.
- Pipette an 80 uL drop of SYBR Green 1 solution onto the slide.
- Pipette an 80 uL drop of sample solutions on top of the SYBR Green 1 drop.
- The drop should rest in the middle of two circles. The solution should not be placed on a previously used portion of the slide.
- Adjust the slide so that the blue light lies on the drop of solution.
- Adjust the smartphone so that it is 9 cm from the drop to the camera.
- Set a timer on the phone camera and close the flap to allow for a dark environment for the picture.
- Pipette the liquid and discard it in the designated area. Move the slide into the next position and repeat.
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
Calibrator Mean Values
|Initial Concentration of 2X Calf Thymus DNA solution (micrograms/mL)
||Final DNA concentration in SYBR Green I solution (µg/mL)
||RAWINTDEN DROP - BACKGROUND
| ||Image 1||Image 2||Image 3||
Images of Our PCR Negative and Positive Controls
PCR Results: PCR concentrations solved
|PCR Product TUBE LABEL
||MEAN (of RAWINTDEN DROP - BACKGROUND)
||PCR Product Concentration (µg/mL)|
(Step 5 calculation)
||Initial PCR Product Concentration (µg/mL)|
(Step 6 calculation)
| G10 +||1561781||1.568894033||12||18.82672839
| G10 -||554868||0.02326170415||12||0.2791404498
| G10 1-1||2543619.33||3.076036223||12||36.91243468
| G10 1-2||1945548.66||2.157985347||12||25.89582416
| G10 1-3||1881616.33||2.059847895||12||24.71817474
| G10 2-1||744948.66||0.3150394577||12||3.780473492
| G10 2-2||1017554.66||0.733495319||12||8.801943827
| G10 2-3||708351.66||0.258862304||12||3.106347648
PCR Results: Summary
- Our positive control PCR result was 18.82672839 μg/mL
- Our negative control PCR result was 0.2791404498 μg/mL
- Patient 54926: Since this patient was positive, the images portrayed a droplet that glowed green. The average quantitative description (μg/mL) of this patient was 29.18 μg/mL.
- Patient 57766: Since this patient was negative, the images portrayed a droplet that was translucent. The average quantitative description (μg/mL) of this patient was 5.23 μg/mL.
- Patient 54926 : The average for this patient was 29.18 ug/mL which is much closer to the positive control, 18.83 ug/mL, than the negative control, 0.279 ug/mL. Therefore, the patient can be concluded as positive because of the quantitative analysis.
- Patient 57766 : The average for this patient was 5.23 ug/mL which is much closer to the negative control, 0.279 ug/mL, than the positive control, 18.83 ug/mL. Therefore, the patient can be concluded as negative because of the quantitative analysis.