LAB 5 WRITE-UP
PCR Reaction Report
The experience with micro pipetting taught us a lot. The pre-lab assignments as well as the tutorials online prepared us well for this lab. They gave us experience with how to work a micro pipetter with confidence. Furthermore, they clearly explained the differences between the first and second stop on the pipette. This allowed us to avoid inconsistencies in our data. It was particularly important in this lab to be able to correctly work a micro pipetter. Our final reactions had an equal amount of liquid so there was not remaining liquid in the DNA and PCR mixture. The tubes were labeled with marker. The tutorials and pre lab materials helped accomplish this. All in all, much was learned about the process of micro pipetting and how to use a fluorimeter.
Smart Phone Camera Settings
- Type of Smartphone: Iphone 5 and Iphone 6
- Flash: Off
- ISO setting: NA
- White Balance: NA
- Exposure: High
- Saturation: High
- Contrast: Low
- Our camera was placed into the cradle and lifted
- Distance between the smart phone cradle and drop = 5.5 cm
Placing Samples onto the Fluorimeter/Taking Fluorimeter Measurements
- Put on gloves and find the smooth side of the glass slide
- Turn on the fluorimeter
- Place slide in the fluorimeter smooth side down
- Set cellphone camera for 3 seconds and place in the cradle
- Adjust height of fluorimeter so that the camera will take a picture of the slide edge-on
- Place an 80uL drop of the SYBR Green I solution on the first two clear circles in the middle of the slide
- Place an 80uL drop of the sample/calibration solution directly on top of the SYBR Green I solution
- Move the slide around until the light hits the drop and focuses the light on the other side
- Adjust the distance between the smartphone and the fluorimeter so that is is greater than 4cm away but not blurry. Record set distance.
- Cover with lightbox and check that the camera is focused
- Press camera button and lower flap before picture it taken
- Discard liquid drop
- Move slide to the next position
- Repeat steps 1-14 with all 5 possible measurement positions.
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
High Calf Thymus DNA
Low Calf Thymus DNA
Zero Calf Thymus DNA
Calibrator Mean Values
|Initial Concentration of 2x Calf Thymus DNA solution (µg/mL)
||Final DNA Concentration in SYBR Green I solution (µg/mL)
||RAWINTDEN Drop - Background
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
|(Step 6 calculation)"
PCR Results: Summary
- Our positive control PCR result was 14.5608 μg/mL
- Our negative control PCR result was 66.9264 μg/mL
- Patient 31801: After combining the SYBR green and the patient's PCR sample, it was evident that the patient was negative. No visible sign of green was detected through the fluorimeter or ImageJ.
- Patient 21190 : The second patient also came up as negative after the test. The fluorimeter and ImageJ did not detect any sign of green within the sample.
- Patient 31801 : Negative
- Patient 21190 : Negative
Based on the results of the PCR product concentrations, we found that patients 31801 and patient 21190 were both negative for the disease. No green appeared in the pictures from the fluorimter in either patient so this is how the conclusion was reached that both patients were negative.