BME100 s2014:T Group3 L5

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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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Contents

OUR TEAM

Name: Kazhan Kader
Name: Kazhan Kader
Name: Maria Morrow
Name: Maria Morrow
Name: Hannah Spehar
Name: Hannah Spehar
Name: Sayer Aldaady
Name: Sayer Aldaady
Name: Jaquelyn Corr
Name: Jaquelyn Corr
Name: Group  3
Name: Group
3


LAB 5 WRITE-UP

Background Information

SYBR Green Dye
SYBR dye is a molecular dye that has fluorescence qualities in the presence of double stranded DNA but not in water or with single stranded DNA. This dye is used to show the presence of dsDNA.


Single-Drop Fluorimeter
The device shines a LED light through a drop of solution sitting on a hydrophobic surface slide that causes the molecules to stick together instead of the plate a spherical shape.


How the Fluorescence Technique Works
SYBR GREEN I dye is used because it fluoresces in the presence of double stranded DNA. When taking a picture of this the fluoresces is picked up the pixels can be analyzed.



Procedure

Smart Phone Camera Settings

  • Type of Smartphone: Droid Razor
    • Flash: Off
    • ISO setting: 800
    • White Balance: Auto
    • Exposure:highest
    • Saturation:highest
    • Contrast: lowest


Calibration


  • Distance between the smart phone cradle and drop = 6cm

Image:SetUpImage.png
Experimental Set Up

Solutions Used for Calibration '

Concentration of 2X Thymus DNA (micrograms/mL) Volume of 2X DNA solution(uL) Volume of the SYBR GREEN I soluion(uL) Final DNA concentration in SYBR green I solution(ug/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. First using the micropipette, pipette 80 microliters of SYBR Green solution onto the slide in the middle on the first row. Then pipette 80 microliters of the 2X calf thymus DNA solution requested in the same place.
  2. The blue LED light was focused on the sample on the plate, and positioned so it is in the middle of the black fiber optic.
  3. The timer was Set on the Smartphone for 10 seconds being placing on the cradle (6cm away), then the lid was closed to the black box and allowing the picture to be taken in complete darkness. This was done twice for every sample
  4. After the pictures were taken, the micropipette was used to remove the solution off the plate. This was repeated 3 times for every concentration.



Data Analysis

Representative Images of Samples
Droplet with no DNA
Image:Negative_Image.png


Droplet with DNA
Image:Positive_Image.png

Image J Values for All Samples

Trial Number Final DNA concentration in SYBR Green I solution Area Mean Pixel Value RawintDen of the Drop Rawintden of the background INTDEN
109463379.54275273306023626924968
10.2511820065.82577805506597017120849
10.510732871.76177019565084407193516
11115140126.8991461120153047314080728
12102036141.8051446924862568513843563
1598864174.7411776990964634817123561
2012254736.61744872955529443934351
20.2512254754.32566573195337106123609
20.512254777.17494574865681828889304
21122547132.7431626726461482615652438
22122547107.1471313057446761812662956
25122547154.521681302748578916327238
3010940830.17533013813670282934353
30.2510940843.45347541334501314304002
30.510940861.55367343934689176265476
3110940893.821102647975291619735636
32109408113.0631236997750156411868413
35109408147.7031615988853369615626192


Fitting a Straight Line

Image:DNA_Graph.png



PCR Results Summary

PCR Product Tube Label Average INTDENS value PCR Product Concentration Corrected PCR Product Concentration
Positive Control 2187513.6670.91042846110.92514153
541172330-8.873821385-106.4858566
542131309.3333-9.072987574-108.8758509
543179070-8.841096901-106.0931628
Negative Control154153-8.962075528-107.5449063
431174222-8.864635224-106.3756227
432174816-8.861751197-106.3410144
433124983-9.103703596-109.2444431


Instructor's summary: You completed 8 PCR reactions in a previous lab. You used the SYBR Green I staining and imaging technique to measure the amount of amplified DNA in each PCR reaction. You used a standard curve (based on known concnetration of claf thymus DNA) to convert INTDEN values into DNA concentration. Your positive control and negative control samples are used to determine the threshold values for determining whether an unknown (patient) sample is truly positive or negative.

Your positive control PCR result was 10.9 μg/mL 

Your negative control PCR result was -107.5 μg/mL


Write-in each patient ID and give both a qualitative (what the images looked like) and a quantitative description (μg/mL) of what you observed
Patient 54174 : -107.2 μg/mL
The images were very dark. The droplets were transparent.
Patient 43184 : -107.3 μg/mL
The images were very dark. The droplets were transparent.

Compare each patient's results to the positive control value and the negative control value. Draw a final conclusion for each patient (positive or negative) and explain why you made that conclusion.
Patient 54174 : Negative because the patients results had an average of -107.2 μg/mL while the negative control had a value of -107.5 μg/mL and the positive control had a value of 10.9 μg/mL. Therefore the patient's results were much more similar to the negative control than the positive control.
Patient 43184 : Negative because the patient results had an average of -107.3 μg/mL while the negative control had a value of -107.5 μg/mL and the positive control had a value of 10.9 μg/mL. Therefore the patient's results were much more similar to the negative control than the positive control.


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