BME100 f2014:Group16 L5

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Owwnotebook icon.png BME 100 Fall 2014 Home
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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
Course Logistics For Instructors
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OUR TEAM

Name: Christopher Saar
Protocol editer
Name: Gurpaul Sidhu
Role(s): Wiki editor, researcher
Picture Source:www. shadyrecords.com/wp-content/uploads/2013/01/royce.jpg
Name: Emily Angeles Mancinas
Role: assistant
Picture Source:http://wallpaperswa.com
Name: Sheania Morgan
Roles: Conductor of experiment
image source: http://www.iemoji.com/view/emoji/814/places/moyai
Name:Leslie Bernardino
Role: assistant/observer
source:www.fanpop.com
Name: Romann Arizmendi
Assistant


LAB 5 WRITE-UP

Procedure

Smart Phone Camera Settings

  • Type of Smartphone:iPhone 5s
    • Flash: NO Flash
    • ISO setting:2000
    • White Balance: Auto
    • Exposure: Zero
    • Saturation:Standard
    • Contrast:Auto


Calibration

  • Distance between the smart phone cradle and drop = 6.5 cm


Solutions Used for Calibration

Init. Conc. 2X Calf Thymus DNA (micrograms/mL) Vol. 2X DNA solution (micro-mL) Vol. SYBR GREEN 1 DYE (micro-mL) Final DNA Concentration STBR GREEN 1 Solution (Micrograms/mL)
5 80 80 2.5
2 80 80 1
1 80 80 0.5
0.5 80 80 0.25
0.25 80 80 0.125
0 80 80 0


Placing Samples onto the Fluorimeter

  1. Place fluorimeter on the table
  2. Place slide in the fluorimeter with the smooth side down hydrophobic side up
  3. Adjust slide to the light shines between the dots on the slide
  4. Place 80 micro-mL of SYBR green 1 on slide between the middle dots
  5. Place 80 micro-mL of calibration on the sphere you already placed to a 160 micro-mL shpere
  6. Adjust the shpere placement for the light and phone distance in the holder
  7. Record Iphone distance from sample
  8. Cover the assembly and make small adjustments
  9. Take a photo on delay and cover
  10. Send photo to be recorded for ImageJ analysis
  11. Remove the 160 micro-mL
  12. Discard the slide
  13. Repeat steps 1-12



Data Analysis

Representative Images of Negative and Positive Samples

POSITIVE CONTROL IMAGE NEGATIVE CONTROL IMAGE
Positive Sample
Negative Sample


Image J Values for All Calibrator Samples

EXCEL Table1 RAW DATA / Drop-Background

DNA Solution Area Pixel Density RAWINTDEN DROP RAWINTDEN BACK RAWINTDEN DROP-BACK
0 58604 61.036 3576976 175112 3401864
0.25 62324 88.828 5536096 175120 5360976
0.5 57291 108.156 6196361 165186 6031175
1 63052 152.1 9590219 189151 9401068
2 54628 195.326 10670272 140838 10529434
5 53200 222.41 11832237 144816 11687421
Positive-1 56188 199.791 11225837 138134 11087703
Positive-2 57469 202.95 11228718 140106 11088612
Positive-3 59569 203.44 11231472 142363 11089109
Negative-1 57504 76.644 4407348 132161 4275187
Negative-2 59222 73.25 4409466 134890 4274576
Negative-3 62109 72.05 4410288 137715 4272573
P1 T1-1 65920 86.368 5693383 169276 5524107
P1 T1-2 66209 83.35 5693737 169817 5523920
P1 T1-3 69148 82.1 5694930 170726 5524204
P1 T2-1 56864 73.951 4205157 142168 4062989
P1 T2-2 59667 74.19 4205869 143593 4062276
P1 T2-3 62540 77.27 4206231 145447 4060784
P1 T3-1 62180 69.03 4292263 149505 4142758
P1 T3-2 63625 67.76 4292430 151453 4140977
P1 T3-3 64252 66.92 4292505 152708 4139797
P2 T1-1 43144 230.772 9956425 177344 9779081
P2 T1-2 43675 260.77 9958551 179669 9778882
P2 T1-3 45197 238.14 9960640 180648 9779992
P2 T2-1 48880 212.619 10392841 146922 10245919
P2 T2-2 49197 205.44 10395760 148476 10247284
P2 T2-3 50275 204.9 10395955 150674 10245281
P2 T3-1 48880 212.619 10392841 121932 10270909
P2 T3-2 49023 206.88 10392996 121968 10271028
P2 T3-3 50014 205.17 10395265 122717 10272548


EXCEL Table 2 Edited to included background-subtractions

Final DNA Concentration in SYBR Green 1 Solution (ug/mL) RAWINTDEN DROP-BACK Standard Deviation
1 2 3 MEAN
0 3401864 3401733 3403177 3402258 799
0.25 5360976 5359811 5358887 5359891 1047
0.5 6031175 6031020 6031474 6031223 231
1 9401068 9399434 9399805 9400102 857
2 10529434 10530655 10530916 10530335 791
5 11687421 11687443 11689630 11688165 1269
Positive 11087703 11088156 11088712 11088190 505
Negative 4275187 4274013 4273439 4274213 891
P1 T1 5524107 5524028 5522487 5523541 913
P1 T2 4062989 4065331 4066390 4064903 1740
P1 T3 4142758 4142815 4144110 4143228 765
P2 T1 9779081 9778693 9778543 9778772 278
P2 T2 10245919 10248449 10248278 10247549 1414
P2 T3 10270909 10272563 10272912 10272128 1070



INITIAL PCR PRODUCT CONCENTRATION = DNA x Total Dilution denominator

PCR Prod Label Mean RAWINTDEN Drop-Background PCR Product Concentration (ug/mL) Intial PCR Product Concentration (ug/mL)
Positive 11088190 8.09 16.18
Negative 4274213 1.27 2.55
P1 T1 5523541 2.52 5.05
P1 T2 4064903 1.06 2.13
P1 T3 4143228 1.14 2.29
P2 T1 9778772 6.78 13.56
P2 T2 10247549 7.25 14.50
P2 T3 10272128 7.27 14.54

Calibration curve
CONTROL CHART

Control



























PATIENT DNA

Patient 1 & 2 Trials




























PCR Results Summary

  • Our positive control PCR result was 11088190 μg/mL
  • Our negative control PCR result was 4274213 μg/mL


Observed results

PATIENT : 37465 PATIENT : 28404
Patient 37436
Patient 28404
NEGATIVE SAMPLE PATIENT: 37465
Negative Sample
Patient 37436
POSITIVE SAMPLE PATIENT: 28404
Positive Sample
Patient 28404



Conclusions

Based upon the graph and the data, Patient 2 is positive and Patient 1 is negative. This is supported by the graph because Patient 2's data values lie above the line of best fit while Patient 1's data value lie below the regression line.



SNP Information & Primer Design

Background: About the Disease SNP

The disease is located on chromosome 21:34370656 on the KCNE2 gene. KCNE stands for potassium voltage-gated channel and joins with KCNH2 gene product, which is a pore forming protein, and alters its function. The mutation is a single nucleotide polymorphism and it is pathogenic in nature. It causes a Cardiovascular disease called Long QT syndrome. This mutation is found in Homo Sapiens by changing the genes that are responsible for encoding the ion channels present within the heart. The normal allele is TTC but it changes to CTC when a person has the disease.

Primer Design and Testing

By inputing the non-disease forward primer and reverse forward primer the PCR database successfully matched the sequence meaning it is found in the healthy human genome. When the disease SNP was inputed with the single nucleotide change, the database could find no matches.
Patient 28404