BME100 f2015:Group11 1030amL5

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Lab Write-Up 1 | Lab Write-Up 2 | Lab Write-Up 3
Lab Write-Up 4 | Lab Write-Up 5 | Lab Write-Up 6
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OUR TEAM

Name: Bernie Silverman
Name: Ashley Hermosillo
Name: Ryan Hryciw
Name: Luis Novelo
Name: Sebastian Kramer
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
5ml 1 2.5 58604 140.108 8210911 480001
5ml 2 2.5 59911 136.987 8207050 446427
5ml 3 2.5 60172 188.288 11028817 420684
2ml 4 1 102423 182.546 18696933 663397
2ml 5 1 90196 194.098 17506839 606940
2ml 6 1 100122 187.709 18793760 669545
1ml 7 0.5 53204 169.577 9022165 560539
1ml 8 0.5 58604 172.399 10103271 597953
1ml 9 0.5 54628 171.86 9388379 551485
.5ml 10 0.25 80526 139.528 11235602 584496
.5ml 11 0.25 83021 136.218 11308946 670847
.5ml 12 0.25 86500 142.208 12300968 587701
.25ml 13 0.125 78723 123.066 968128 604677
.25ml 14 0.125 83330 106.742 8894813 1242142
.25ml 15 0.125 78723 107.274 8444908 1184645
0ml 16 0 85054 83.392 7262906 821217
0ml 17 0 83330 83.744 6978426 783993
0ml 18 0 97972 88.35 8655810 896692
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)"
negative 9363938 13.8588 12 166.3056
1=1 8239334 7.2657 12 87.1884
1=2 7817477 4.792 12 57.504
1=3 5966138 -6.061 12 (-72.732)
2=1 6583928 -2.45 12 (-29.4)
2=2 7824816 4.836 12 58.032
2=3 7038079 0.223 12 2.676
positive 17872824 1006.774 12 12081.288

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 1 96670 108.587 10497142 877995 9619147
negative n 2 89338 102.517 9158627 720565 8438062
negative n 3 93357 107.004 9989540 880811 9108729
1_1 4 87218 100.252 8743812 666542 8077270
1_1 5 84636 101.662 8600843 669491 7931352
1_1 6 88166 102.453 9032895 631497 8401398
1_2 7 86088 101.123 8705476 720871 7984605
1_2 8 86325 100.495 8675211 718113 7957098
1_2 9 81988 102.383 8394203 743854 7650349
1_3 10 75812 84.763 6426086 599890 5826196
1_3 11 69934 84.077 5879847 558317 5321530
1_3 12 77062 86.93 6698979 592900 6106079
2_1 13 81541 87.041 7097449 768089 6329360
2_1 14 81336 88.105 7166089 807823 6358266
2_1 15 85111 89.231 7594505 756010 6838495
2_2 16 83328 102.712 8558802 755442 7803360
2_2 17 82273 106.212 8738353 614264 8124089
2_2 18 82292 103.345 8504506 658235 7846271
2_3 19 68930 105.298 7258196 390891 6867305
2_3 20 72988 107.921 7876923 482068 7394855
2_3 21 69672 109.464 7626573 417721 7208852
Positive P 22 85083 210.537 17913085 427129 17485956
Positive P 23 84721 214.511 18173620 424455 17749165
Positive P 24 88124 211.802 18664807 405016 18259791


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.