# BME100 f2017:Group10 W1030 L5

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# OUR TEAM

 Name: Sarah Desmond Name: Eddie Aparicio Name: Patrick Mullane Name: Adrienne Parkinson Name: Leslie Olivares

# LAB 5 WRITE-UP

## PCR Reaction Report

While we were micropipetting the samples to set up the reaction, we felt that we were well prepared from watching the pre-lab videos and reading the documents. We moved the samples with ease while using the micropipetting technique. We understood the difference between the first and second stop on the pipettor with help from the pre-lab materials. The final reactions had exactly the same amount of liquid because we pipetted all of the liquid from the samples that were given to us. There was no liquid left in the tubes that the DNA samples and PCR reaction mix, which means that we pipetted the same amount of each. We did not have to change our labelling scheme because our system allowed for clear differences between the samples.

## Fluorimeter Procedure

Imaging set-up

While we were setting up our device to capture the images from the fluorimeter, we stared with turning on the fluorimeter and placed it on the table. Then, we placed the slide in the fluorimeter with the sooth side down. After that, we adjusted the height of the fluorimeter to the height of the camera in order to capture a proper image using a camera timer set for 3 seconds on a smartphone.

Placing Samples onto the Fluorimeter

1. The first step is to identify two clear circles on the middle of the slide where we will then place 80 uL drop of SYBR Green 1 solution.
2. Step two is to the place 80 uL drop of sample/calculation that will mix with the SYBR Green Solution 1.
3. Then, make sure that the blue light lights up the center of the drop by moving the slide accordingly.
4. Making certain that the phone is more than 4 centimeters away from the drop, record the distance and ensure that you are able to have a clear, consise view of the drop thorugh the lens.
5. After that, place the light box with the open side facing the lab group, and double check that the drop is focused with the camera an additional time before the picture is taken.
6. After the picture is taken, remove the drop from the slide and discard the waste into a liquid waste container. Lastly, move the slide to the next row of two circles for the next drop to be analyzed.
7. Repeat steps 1-6 for all samples.

## Data Collection and Analysis

Images of High, Low, and Zero Calf Thymus DNA

5 μg/mL sample




0.5 μg/mL sample

zero DNA

Calibrator Mean Values

Initial Concentration of 2X Calf Thymus DNA solution (micrograms/mL) Initial Concentration of 2X Calf Thymus DNA solution (micrograms/mL) Final DNA concentration in SYBR Green I solution (µg/mL) Sample Number RAWINTDEN DROP - BACKGROUND MEAN Standard Deviation
Image 1 Image 2 Image 3
5 5 2.5 C-1 135097 119824 111796 122239 11836.73599
2 2 1 C-2 107735 111997 107583 109105 2505.698306
1 1 0.5 C-3 79634 83274 77511 80140 2914.58682
0.5 0.5 0.25 C-4 77443 76321 79551 77772 1639.890647
0.25 0.25 0.125 C-5 69680 79559 68328 72522 6131.311795
0 0 0 C-6 C-6 0 0 0 0

Calibration curves

Images of Our PCR Negative and Positive Controls

Negative Control

Positive Control

PCR Results: PCR concentrations solved

PCR Product TUBE LABEL MEAN (of RAWINTDEN DROP - BACKGROUND) "PCR Product Concentration (µg /mL)" Total Dilution "Initial PCR Product Concentration (µg /mL)"
G10+ 3477766 322.2470889 12 3866.965067
G10- 1135421.667 100.4971757 12 1205.966108
G10 1-1 2557789 235.1527975 12 2821.83357
G10 1-2 2555368.667 234.9236644 12 2819.083973
G10 1-3 3234299 299.1980498 12 3590.376598
G10 2-1 1110961.667 98.18154568 12 1178.178548
G10 2-2 687185.3333 58.06260847 12 696.7513017
G10 2-3 538257 43.96355202 12 527.5626243

PCR Results: Summary

• Our positive control PCR result was 3866.965067 μg/mL
• Our negative control PCR result was 1205.966108 μg/mL

Observed results

• Patient 72874: Qualitatively, the images taken for this patient in the fluorimeter showed a bright green color within the droplet of SYBR Green I and sample. The average in concentration for patient 1 was closer to the control positive control.
• Patient 64687: The images for this patient showed a very faint/dark green color within the SYBr Green 1 solution droplet. The average in concentration for this patient was also closer to its negative control value.

Conclusions

• Patient 72874: This patient tested positive because its concentration tested closer to the positive control value, and illuminated green within the SYBR Green 1 solution.
• Patient 64687: This patient tested negative because its concentration was closer to the negative control value and did not illuminate green at all in the SYBR Green 1 solution.