BME100 f2016:Group15 W1030AM L5

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Group still avoiding freshman 15

Name: Kelsey Boos
Name: Myong Chung
Name: Ashwin Limaye
Name: Kyle Caalim
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Name: student
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Name: student


LAB 5 WRITE-UP

PCR Reaction Report

Summary: Through the experiment, it was helpful to have read the pre-lab reading beforehand as well as having it while doing the trials. This was because the process of pipetting and using the samples was known before the experiment and there was less chances for error to occur during the experiment. The pre-lab made the difference between the first stop and the second stop very clear, so it may have helped measurements be more accurate overall. In general, the final amounts of each mixture were similar, but at times weren't exactly the same. This may have been due to a slight mistake in pipetting or there may have also been a bubble that formed in the pipette.In general, there was no liquid left in the tubes for the DNA samples, but there was still leftover PCR Reaction mix that could have been used. We did not have to change our labeling scheme and it stayed the same through both of the experiments.

Fluorimeter Procedure

Imaging set-up


To set up the device, we used a smartphone to help detect fluorescence for DNA samples. First, we used a Samsung galaxy s7 and changed the camera settings. We did this by inactivating the flash, setting iso to 800, setting white balance to auto, setting exposure and saturation to highest settings, and setting contrast to lowest settings. Then we also installed image J on the computer for analysis.

Placing Samples onto the Fluorimeter

Step One: Place a 160 microliter drop of water (H2O) in the middle of the first two rows of the slide using the pipettor so that the drop is pinned and looks like a beach ball.

Step Two: Turn on the excitation light using the switch for the Blue LED.

Step Three: Turn on the camera of your smart phone and check the settings on the smartphone

Step Four: Place your smart phone on the cradle at a right angle from the slide.

Step Five: Adjust the distance between the smartphone on its cradle and the first two rows of the slide so that it is as close as possible without making the image blurry. It should be at least 4 cm away from the drop.

Step Six: Record the distance between your smart phone cradle and drop using the ruler provided in lab. Be careful not to move the camera, cradle, or fluorimeter very much. The light collected will change slightly if there is a significant difference from one image to the next in these distances.

Step Seven: Place a 80 microliter drop of SYBR GREEN I in the middle of the first two rows of the slide using the pipettor so that the drop is pinned and looks like a beach ball.

Step Eight: Align the drop by moving the slide so that the blue LED light is focused by the drop to the middle of the black fiber optic fitting on the other side of the drop.

Step Nine: Use the timer on your camera so that you can take a picture after covering the fluorimeter and camera with the light box.

Step Ten: Take three images​ of the drop being careful to check that drop is focused.

Step Eleven: Use the pipettor to remove the 160 microliter drop from the surface and move the slide to the next position.

Step Twelve: repeat these steps for the other concentrations.


Data Collection and Analysis

Images of High, Low, and Zero Calf Thymus DNA

High: HighDrop2.jpg

Low: LowDrop1.jpg

Water: WaterDrop1.jpg


Calibrator Mean Values

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 (Image 1) RAWINTDEN DROP - BACKGROUND (Image 2) RAWINTDEN DROP - BACKGROUND (Image 3) Mean Standard Deviation
5 2.5 C-1 6765713 6526629 6590974 6627672 123716.8516
2 1 C-2 5321069 5531213 5460496 5437463 106927.7662
1 0.5 C-3 7834815 7923275 7886765 7881567 44454.01032
0.5 0.25 C-4 4703935 4619340 4567450 4630216 68892.47824
0.25 0.125 C-5 2547532 2546434 2640062 2578009 53741.98993
0 0 C-6 159191 110301 111320 126918 27937.14106


Calibration curves
DOT PLOT 1.jpg DOT PLOT 2.jpg


Images of Our PCR Negative and Positive Controls

Positive Control: PControl1.jpg

Negative Control: NControl1.jpg

PCR Results: PCR concentrations solved

PCR Product TUBE LABEL MEAN (of RAWINTDEN DROP - BACKGROUND) PCR Product Concentration (µg /mL)(Step 5 calculation) Total Dilution Initial PCR Product Concentration (µg /mL)(Step 6 calculation)
G2 P 2864263 0.5099928352 12 6.119914023
G2 N 171418.666 -2.246244968 12 -26.95493962
G2 1-1 290003 -2.124868987 12 -25.49842784
G2 1-2 106285.333 -2.312911635 12 -27.75493962
G2 1-3 142526 -2.27581781 12 -27.30981372
G2 2-1 164136 -2.253699079 12 -27.04438895
G2 2-2 192306 -2.224865916 12 -26.69839099
G2 2-3 351028.666 -2.062406688 12 -24.74888025


PCR Results: Summary

  • Our positive control PCR result was 6.1199 μg/mL
  • Our negative control PCR result was -27.3098 μg/mL


Observed results

  • Patient 10842 : Negative
  • Patient 19457 : Negative


Conclusions

  • Patient 10842 : NO
  • Patient 19457 : NO