BME100 s2014:W Group1 L5

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Owwnotebook icon.png BME 100 Spring 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
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OUR TEAM

Name: Maria Jose Quezada
Role(s): Protocols
Name: Danielle Eldred
Role(s): Open PCR and Machine Testing
Name: John Tobey
Role(s): Research and Development
Name: David Kish
Role(s): Open PCR and Machine Testing
Name: Khalid Al-Qahtani
Role(s): Research and Development

LAB 5 WRITE-UP

Background Information

SYBR Green Dye
A small molecular dye that fluoresces very well in the presence of dsDNA but fluoresces very weakly in water or with single strands of DNA.


Single-Drop Fluorimeter
This instrument that detects fluorescence; quality is related to the amount of florescent material and, less directly, proportional to the amount of the molecule being detected.


How the Fluorescence Technique Works
A very sensitive detection technique that uses the generation of light by a molecule due to excitation by shining light of a shorter wavelength.

Procedure

Smart Phone Camera Settings

  • Type of Smartphone: Apple iPhone 5
    • Flash: Inactivate
    • ISO setting: 2000
    • White Balance: N/A
    • Exposure: N/A
    • Saturation: N/A
    • Contrast: N/A


Calibration

1. Turn on the fluorimeter and put it on top of the table.

2. Using plastic plates,or other kind of structure adjust the height of the fluorimeter box.

3. Place the cradle with your phone in camera mode on.

4. Make sure that the distance between the fluorimeter and your phone is larger than 4cm in order to get a clear picture.

5. Cover the fluorimeter and the cradle with the light box keeping one flap up.

Flour.jpg

  • Distance between the smart phone cradle and drop = 5 cm


Solutions Used for Calibration

Initial Concentration of 2X Calf Thymus DNA solution (micrograms/mL) Volume of the 2X DNA solution (μL) Volume of the SYBR Green 1 Dye solution (μL) Final DNA concentration in SYBR Green 1 Solution (μg/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 slide on Fluorimeter so that the blue light comes between the second and third hole

2. Apply tip to Micro-pipette

3. Insert tip of Micro-pipette into sample

4. Press button to absorb sample

5. Remove tip from sample

6. Press button while over second and third holes of slide to eject sample

7. Eject tip of Micro-pipette

8. Apply tip to Micro-pipette

9. Insert tip into SYBR 1 Green

10. Press button to absorb SYBR 1 Green

11. Remove tip from SYBR 1 Green

12. Press Button while over sample droplet to eject SYBR 1 Green to eject into sample

13. Eject tip

14. Repeat this process for all following samples


Data Analysis

Representative Images of Samples

WithoutDNA.jpg

  • Image J analysis of sample without DNA

WithDNA.jpg

  • Image J analysis of sample with DNA


Image J Values for All Samples

[Instructions: See worksheet page 8. To save time on typing a new Wiki table from scratch, use THIS TOOL to auto-generate a Wiki table: Excel-to-Wiki Converter. Copy the headers and values from the Excel spreadsheet you made, paste them into the form field, click submit, copy the Wiki code that the tool generated, and replace TABLE GOES HERE (below) with your auto-generated code.]

DNA concentration in SYBR Green I solution Area Mean Pixel Value Rawintden of the drop Rawintden of the Background
0 123524 12.446 1537377 499658
0 114081 8.257 941977 269357
0 151176 18.676 2823290 710551
0.25 158734 76.606 12160018 787735
0.25 134552 47.007 6324891 326289
0.25 133871 48.121 6442047 428723
0.5 166932 56.991 9513592 617318
0.5 147194 32.221 4742804 394599
0.5 146386 30.238 4426407 342671
1 118008 81.262 9589623 1098336
1 183212 58.828 10777923 745412
1 247655 123.415 30564391 845749
2 197697 114.558 22647709 768839
2 183426 119.708 21957473 544822
2 277605 124.475 34555021 1129608
5 215236 177.602 38226246 896635
5 256884 174.248 44622241 794800


Fitting a Straight Line


Calibrationgraph1.png

PCR Results Summary

Our positive control PCR result was: 0.2559 μg/mL

Our negative control PCR result was: 0.0475 μg/mL


Patient 10939 : Patient 10939 exhibited a green fluorescence. This, along with PCR concentrations similar to the positive control, indicate that this patient had the screened-for DNA.

Patient 57695 : Patient 57695 exhibited no fluorescence. This, along with PCR concentrations similar to the negative control, indicate that this patient did not have to screened-for DNA.


Finalresults.jpg

  • Final Concentration Results