BME100 f2014:Group16 L5

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BME 100 Fall 2014 Home
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Lab Write-Up 1 | Lab Write-Up 2 | Lab Write-Up 3
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Contents

OUR TEAM

Name: Christopher SaarProtocol editer
Name: Christopher Saar
Protocol editer
Name: Gurpaul SidhuRole(s): Wiki editor, researcherPicture Source:www. shadyrecords.com/wp-content/uploads/2013/01/royce.jpg
Name: Gurpaul Sidhu
Role(s): Wiki editor, researcher
Picture Source:www. shadyrecords.com/wp-content/uploads/2013/01/royce.jpg
Name: Emily Angeles MancinasRole: assistantPicture Source:http://wallpaperswa.com
Name: Emily Angeles Mancinas
Role: assistant
Picture Source:http://wallpaperswa.com
Name: Sheania MorganRoles: Conductor of experiment  image source: http://www.iemoji.com/view/emoji/814/places/moyai
Name: Sheania Morgan
Roles: Conductor of experiment
image source: http://www.iemoji.com/view/emoji/814/places/moyai
Name:Leslie BernardinoRole: assistant/observersource:www.fanpop.com
Name:Leslie Bernardino
Role: assistant/observer
source:www.fanpop.com
Name: Romann Arizmendi  Assistant
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 IMAGENEGATIVE CONTROL IMAGE
Positive Sample
Positive Sample
Negative 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
05860461.03635769761751123401864
0.256232488.82855360961751205360976
0.557291108.15661963611651866031175
163052152.195902191891519401068
254628195.3261067027214083810529434
553200222.411183223714481611687421
Positive-156188199.7911122583713813411087703
Positive-257469202.951122871814010611088612
Positive-359569203.441123147214236311089109
Negative-15750476.64444073481321614275187
Negative-25922273.2544094661348904274576
Negative-36210972.0544102881377154272573
P1 T1-16592086.36856933831692765524107
P1 T1-26620983.3556937371698175523920
P1 T1-36914882.156949301707265524204
P1 T2-15686473.95142051571421684062989
P1 T2-25966774.1942058691435934062276
P1 T2-36254077.2742062311454474060784
P1 T3-16218069.0342922631495054142758
P1 T3-26362567.7642924301514534140977
P1 T3-36425266.9242925051527084139797
P2 T1-143144230.77299564251773449779081
P2 T1-243675260.7799585511796699778882
P2 T1-345197238.1499606401806489779992
P2 T2-148880212.6191039284114692210245919
P2 T2-249197205.441039576014847610247284
P2 T2-350275204.91039595515067410245281
P2 T3-148880212.6191039284112193210270909
P2 T3-249023206.881039299612196810271028
P2 T3-350014205.171039526512271710272548


EXCEL Table 2 Edited to included background-subtractions

Final DNA Concentration in SYBR Green 1 Solution (ug/mL) RAWINTDEN DROP-BACK Standard Deviation
123MEAN
03401864340173334031773402258799
0.2553609765359811535888753598911047
0.56031175603102060314746031223231
19401068939943493998059400102857
210529434105306551053091610530335791
5116874211168744311689630116881651269
Positive11087703110881561108871211088190505
Negative4275187427401342734394274213891
P1 T15524107552402855224875523541913
P1 T240629894065331406639040649031740
P1 T34142758414281541441104143228765
P2 T19779081977869397785439778772278
P2 T2102459191024844910248278102475491414
P2 T3102709091027256310272912102721281070



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)
Positive110881908.0916.18
Negative42742131.272.55
P1 T155235412.525.05
P1 T240649031.062.13
P1 T341432281.142.29
P2 T197787726.7813.56
P2 T2102475497.2514.50
P2 T3102721287.2714.54

Calibration curve
CONTROL CHART

Control
Control



























PATIENT DNA

Patient 1 & 2 Trials
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 : 37465PATIENT : 28404
Patient 37436
Patient 37436
Patient 28404
Patient 28404
NEGATIVE SAMPLEPATIENT: 37465
Negative Sample
Negative Sample
Patient 37436
Patient 37436
POSITIVE SAMPLEPATIENT: 28404
Positive Sample
Positive Sample
Patient 28404
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
Patient 28404


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