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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OUR TEAM

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

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
Negative Image.png


Droplet with DNA
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
1 0 94633 79.542 7527330 602362 6924968
1 0.25 118200 65.825 7780550 659701 7120849
1 0.5 107328 71.761 7701956 508440 7193516
1 1 115140 126.899 14611201 530473 14080728
1 2 102036 141.805 14469248 625685 13843563
1 5 98864 174.741 17769909 646348 17123561
2 0 122547 36.617 4487295 552944 3934351
2 0.25 122547 54.325 6657319 533710 6123609
2 0.5 122547 77.174 9457486 568182 8889304
2 1 122547 132.743 16267264 614826 15652438
2 2 122547 107.147 13130574 467618 12662956
2 5 122547 154.52 16813027 485789 16327238
3 0 109408 30.175 3301381 367028 2934353
3 0.25 109408 43.453 4754133 450131 4304002
3 0.5 109408 61.553 6734393 468917 6265476
3 1 109408 93.821 10264797 529161 9735636
3 2 109408 113.063 12369977 501564 11868413
3 5 109408 147.703 16159888 533696 15626192


Fitting a Straight Line

DNA Graph.png



PCR Results Summary

PCR Product Tube Label Average INTDENS value PCR Product Concentration Corrected PCR Product Concentration
Positive Control 2187513.667 0.910428461 10.92514153
541 172330 -8.873821385 -106.4858566
542 131309.3333 -9.072987574 -108.8758509
543 179070 -8.841096901 -106.0931628
Negative Control 154153 -8.962075528 -107.5449063
431 174222 -8.864635224 -106.3756227
432 174816 -8.861751197 -106.3410144
433 124983 -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.