BME100 f2014:Group14 L5

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OUR TEAM

Name: Fatima Sanchez Garcia
Lemlem Brook
Claudia Czekaj
Name: Ciaran McGirr
Taylor Brown
Brianna Steele


LAB 5 WRITE-UP

Procedure

Smart Phone Camera Settings

  • Type of Smartphone: iPhone 5C
    • Flash: no flash
    • ISO setting: 800
    • White Balance: not able to adjust
    • Exposure: not able to adjust
    • Saturation: not able to adjust
    • Contrast: not able to adjust


Calibration

  • To set up the camera, we placed it in the phone cradle and set up a timer for three seconds. We placed the phone more than four centimeters away from the fluorimeter and made sure it was even with the drop.
  • Distance between the smart phone cradle and drop = 4 centimeters


Solutions Used for Calibration

Initial Concentration of 2X Calf Thymus DNA Solution (µg/mL) Volume of 2X DNA Solution (µL) Volume of SYBR Green I Dye Solution (µL) Final DNA Concentration in SYBR Green I Solution (µg/mL)
5 80 80 2.5
2 80 80 1
1 80 80 .5
.5 80 80 .25
.25 80 80 .125
0 80 80 0


Solutions Used for PCR Samples

PCR Product Tube Label Volume of the Diluted PCR Product solution(μL) Volume of SYBR Green I Dye Solution (µL) Dilution 1 Dilution 2 Total Dilution Simplified Fraction
14 1-1 80 80 0.166 .5 0.0833
14 1-2 80 80 0.166 .5 0.0833
14 1-3 80 80 0.166 .5 0.0833
14 Pos. 80 80 0.166 .5 0.0833
14 2-1 80 80 0.166 .5 0.0833
14 2-2 80 80 0.166 .5 0.0833
14 2-3 80 80 0.166 .5 0.0833
14 Neg. 80 80 0.166 .5 0.0833



Placing Samples onto the Fluorimeter

  1. Step One: With the fluorimeter on, put the smooth-side of the slide down into the fluorimeter.
  2. Step Two: Open the camera application and set a three second timer with no flash.
  3. Step Three: Place 80 μL of SYBR Green in between the first two circles in the middle row of the slide.
  4. Step Four: Add 80 μL of the Sample Solution to the SYBR Green on the slide using the micropipette.
  5. Step Five: Focus the fluorimeter light on the center of the drop on the slide.
  6. Step Six: Place the camera approximately 4 cm away from the fluorimeter.
  7. Step Seven: Use the box to cover the fluorimeter and the phone.
  8. Step Eight: Make sure the drop is focused on the camera, press the camera button and lower the flap of the box.
  9. Step Nine: Wait approximately 5 seconds to ensure that the picture is taken without disturbance from outside light.
  10. Step Ten: Reopen the flap and take another two pictures.
  11. Step Eleven: Open the flap and remove the 160 μL drop of SYBR Green and Sample Solution from the slide using the micropipette and discard in the waste container.
  12. Step Twelve: Adjust the slide so the light focuses on the next set of circles.
  13. Steps Thirteen: Repeat steps Two through Eleven until all solutions have been placed on the fluorimeter and photographed.


Data Analysis

Representative Images of Negative and Positive Samples
This is the positive sample in the flourimeter.
Group14 positive l5.JPG

This is the negative sample in the flourimeter.
Negative test l5 imagej.JPG


Image J Values for All Calibrator Samples

Table 1 Description: Table 1 shows all the data taken during the lab and all of the date extracted from ImageJ.

Final DNA concentration in SYBR Green I solution (ug/mL) AREA Mean Pixel Value RAWINTDEN of the drop RAWINTDEN of the background RAWINTDEN drop-background
2.5 #1 14176 105.51 1495714 21398 1474316
2.5 #2 14590 99.548 1452403 25555 1426848
2.5 #3 15092 101.881 1537586 28563 1509023
1 #1 15402 86.36 1330121 26902 1303219
1 #2 15272 81.5 1244661 29992 1214669
1 #3 15200 82.865 1259552 27209 1232343
.5 #1 11770 91.15 1072830 16946 1055884
.5 #2 10309 95.702 986588 17298 969290
.5 #3 10748 101.886 1094851 15828 1079023
.25 #1 11398 83.283 949255 23992 925263
.25 #2 10646 79.226 843438 16114 827324
.25 #3 10501 82.391 865190 14462 850728
.125 #1 14750 59.167 872719 26834 845885
.125 #2 13853 60.16 833390 30909 802481
.125 #3 13488 63.964 862752 28086 834666
H2O #1 15668 36.41 570466 29545 540921
H2O #2 16158 37.729 609629 32081 577548
H20 #3 16926 36.272 613933 31559 582374
tube 1-1 #1 14774 98.509 1455374 28109 1427265
tube 1-1 #2 15380 96.776 1488419 25996 1462423
tube 1-1 #3 14520 98.487 1430028 21638 1408390
tube 1-2 #1 12100 107.7 1303256 24759 1278497
tube 1-2 #2 12697 105.207 1335808 22155 1313653
tube 1-2 #3 13792 100.782 1389992 26877 1363115
tube 1-3 #1 12092 85.422 1032918 25923 1006995
tube 1-3 #2 10888 93.493 1017951 20411 997540
tube 1-3 #3 12052 86.662 1044452 23297 1021155
positive #1 16484 87.126 1436182 31255 1404927
positive #2 16390 90.677 1486197 29471 1456726
positive #3 16723 89.235 1492281 32215 1460066
tube 2-1 #1 14916 41.782 623226 28122 595104
tube 2-1 #2 15817 39.806 629618 30172 599446
tube 2-1 #3 15036 41.319 621275 27440 593835
tube 2-2 #1 13192 14.357 189394 26457 162937
tube 2-2 #2 12648 14.322 181147 24540 156607
tube 2-2 #3 12842 15.617 200552 24212 176340
tube 2-3 #1 14256 27.649 394162 28295 365867
tube 2-3 #2 14774 22.203 328032 28679 299353
tube 2-3 #3 14520 21.113 306566 25369 281197
negative #1 16308 42.543 693793 33092 660701
negative #2 16353 45.996 752173 35035 717138
negative #3 15913 45.021 716418 32452 683966


Table 2 Description: Table 2 includes the Mean and Standard Deviation calculations of the Final DNA Concentrations. The Mean calculations and Concentrations were used to create the Calibration Curve.

Final DNA Concentration in SYBR Green I Solution (ug/mL) Mean Standard Deviation
0 566947.7 22668.54875
0.125 826677.3 21353.80295
0.25 867771.7 51145.64361
0.5 1031399 63510.8986
1 1250077 46863.06319
2.5 1470062 41252.30801


Table 3 Description: The data from Table 3 were calculated using the Calibration Curve to find the unknown concentrations of SYBR Green Solution. This information was then used to determine if the patients were positive for the gene.

Final DNA concentration in SYBR Green I Solution (ug/mL) PCR Product Concentration (ug/mL) Initial PCR Product Concentration (ug/mL)
tube 1-1 1.231454625 14.7774555
tube 1-2 1.044726016 12.53671219
tube 1-3 0.538390938 6.460691251
tube 2-1 -0.135563207 -1.62675848
tube 2-2 -0.839582349 -10.07498819
tube 2-3 -0.594179169 -7.130150025
positive 1.244331765 14.93198118
negative 0.013367366 0.160408391

Calibration Curve
Calibration Curve Description: The calibration curve show the relationship between concentration of the solution and the Mean RAWINTDEN drop background of known concentrations. The concentration data from 2.5 μg/mL was omitted from the line of best fit because it was an outlier.

DNA Concentrations vs INTDEN.jpg


PCR Results Summary

  • Our positive control PCR result was 14.93198118 μg/mL
  • Our negative control PCR result was 0.160408391 μg/mL

Observed results

  • Patient 1-22157 : The images were more florescent than patient 49284. They had a very strong green color.
  • Patient 2-49384 : The images had more of a darker color. There was no florescence in the images from patient 49284.

Conclusions

  • Patient 1-22157 : The SYBR Green fluoresces very well in the presence of the dsDNA.
  • Patient 2-49384 : There was no florescence in the drop because there was no dsDNA present.




SNP Information & Primer Design

Background: About the Disease SNP

Single Nucleotide Polymorphism (also known as SNP) is a variation within the DNA sequence. This variation is common within the population, meaning it is prevalent in 1% of the population. SNPs are differences in only one DNA nucleotide. Nucleotides are the building blocks for DNA and are made up of a nitrogenous base, sugar, and phosphate. SNPs are typically found in non-coding regions of DNA leading to no visible results of this variation. While most SNPs have no effect on a person's health, they can in certain cases predict disease, response to drugs, and other clinical conditions.

Primer Design and Testing

When testing the DNA sequence of the rs16991654 gene, we looked at both the diseased and non-diseased primers. The forward primer, was the DNA sequence of the top strand read from left to right while the reverse primer was the DNA sequence of the lower strand of DNA read from right to left. When we tested the primers for the non-diseased DNA we found that it returned the sequence of the normal human DNA. When we tested the diseased DNA, no matches were returned. This is because the variation in this diseased DNA is not meant to be in a healthy genome; its a SNP.

BME Forward Primer.PNG

Sequence used for Forward Primer. To create diseased primer, the Thymine at the end was changed into a Cytosine.

BME Reverse Primer.PNG

Sequence used for Reverse Primer. Sequence is to be read from right to left.

BME Normal Primer.PNG

Results for Non-Diseased Primer

BME Disease Primer.PNG

Results for Diseased Primer