BME100 f2015:Group11 1030amL5

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Contents

OUR TEAM

Name: Bernie Silverman
Name: Bernie Silverman
Name: Ashley Hermosillo
Name: Ashley Hermosillo
Name: Ryan Hryciw
Name: Ryan Hryciw
Name: Luis Novelo
Name: Luis Novelo
Name: Sebastian Kramer
Name: Sebastian Kramer
Name: Raegan Barry
Name: Raegan Barry


LAB 5 WRITE-UP

PCR Reaction Report

When pipetting the samples, our group did not run into any issues. This was in part due to the pre-lab reading, which explained in detail on how we were to go about pipetting the samples without making errors. The pre-lab reading also helped us understand the PCR process and allowed us to calibrate the PCR device, which included the steps and temperatures of each step of the PCR process. The first stop of the pipettor was to vacate air and to prepare for the correct amount of liquid. The second stop was to make sure that all of the liquid taken up by the pipettor has left into the desired tube. The final reactions had the same amount of liquid because the micropipettor was very specific as to how much liquid was put into each tube. No liquids were left behind in the tubes where the DNA samples and PCR reactions mixed. Our labeling scheme followed that of the PDF file for the lab; nothing needed to be changed.

Fluorimeter Procedure

Smart Phone Camera Settings

  • Type of Smartphone:Samsung Galaxy S6
    • Flash:No
    • ISO setting:0
    • White Balance:0
    • Exposure:0
    • Saturation:0
    • Contrast:0


Camera set-up

  • Distance between the smart phone cradle and drop = 5cm


Placing Samples onto the Fluorimeter

  1. Place 80 microliters of SYBRI onto the glass slide
  2. Add the specified concentration of the 2X Calf Thymus DNA Solution to the SYBRI.
  3. Align the blob of liquid with the blue LED light so that it is focused to the middle of the black optic fiber on the other side of the blob.
  4. Repeat steps 1-3 for each of the 6 concentrations.


Data Collection and Analysis

Images of High, Low, and Zero Calf Thymus DNA


High Concentration High Concentration

Low Concentration Low Concentration

Zero Concentration Zero Concentration

Calibrator Mean Values


sample number image number final DNA concentration in sypr green 1 solution (micrograms/mL) AREA Mean Pixel Value RAWINTDEN of the drop RAW INTDEN of the background
5ml12.558604140.1088210911480001
5ml22.559911136.9878207050446427
5ml32.560172188.28811028817420684
2ml41102423182.54618696933663397
2ml5190196194.09817506839606940
2ml61100122187.70918793760669545
1ml70.553204169.5779022165560539
1ml80.558604172.39910103271597953
1ml90.554628171.869388379551485
.5ml100.2580526139.52811235602584496
.5ml110.2583021136.21811308946670847
.5ml120.2586500142.20812300968587701
.25ml130.12578723123.066968128604677
.25ml140.12583330106.74288948131242142
.25ml150.12578723107.27484449081184645
0ml1608505483.3927262906821217
0ml1708333083.7446978426783993
0ml1809797288.358655810896692
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)"
negative936393813.858812166.3056
1=182393347.26571287.1884
1=278174774.7921257.504
1=35966138-6.06112(-72.732)
2=16583928-2.4512(-29.4)
2=278248164.8361258.032
2=370380790.223122.676
positive178728241006.7741212081.288

Calibration curves
Calibration Curve 1 Calibration Curve 2

Images of Our PCR Negative and Positive Controls


Negative Control Picture Negative Control

Positive Control Picture Positive Control


PCR Results: PCR concentrations solved

PCR product tube label image number AREA Mean Pixels RAWINTDEN of the drop RAWINTDEN of the background RAWINTDEN drop - background
negative n 196670108.587104971428779959619147
negative n 289338102.51791586277205658438062
negative n 393357107.00499895408808119108729
1_1487218100.25287438126665428077270
1_1584636101.66286008436694917931352
1_1688166102.45390328956314978401398
1_2786088101.12387054767208717984605
1_2886325100.49586752117181137957098
1_2981988102.38383942037438547650349
1_3107581284.76364260865998905826196
1_3116993484.07758798475583175321530
1_3127706286.9366989795929006106079
2_1138154187.04170974497680896329360
2_1148133688.10571660898078236358266
2_1158511189.23175945057560106838495
2_21683328102.71285588027554427803360
2_21782273106.21287383536142648124089
2_21882292103.34585045066582357846271
2_31968930105.29872581963908916867305
2_32072988107.92178769234820687394855
2_32169672109.46476265734177217208852
Positive P 2285083210.5371791308542712917485956
Positive P 2384721214.5111817362042445517749165
Positive P 2488124211.8021866480740501618259791


PCR Results: Summary

  • Our positive control PCR result was 2.5 μg/mL
  • Our negative control PCR result was 0 μg/mL


Observed results

  • Patient 79685: The water droplet was all blue when viewing them in the Florimeter and contained no amount of green disease.
  • Patient 90772: The water droplet was all blue when viewing them in the Florimeter and contained no amount of green disease.


Conclusions

  • Patient 79685: This patient's results came back negative which means that they did not have the disease.
  • Patient 90772: This patient's results also came back negative which means that they also did not have the disease.



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