BME100 s2014:W Group1 L5

From OpenWetWare

Jump to: navigation, search
BME 100 Spring 2014 Home
People
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
Photos
Wiki Editing Help
Image:BME494_Asu_logo.png


Contents

OUR TEAM

Name: Maria Jose QuezadaRole(s): Protocols
Name: Maria Jose Quezada
Role(s): Protocols
Name: Danielle EldredRole(s): Open PCR and Machine Testing
Name: Danielle Eldred
Role(s): Open PCR and Machine Testing
Name: John TobeyRole(s): Research and Development
Name: John Tobey
Role(s): Research and Development
Name: David KishRole(s): Open PCR and Machine Testing
Name: David Kish
Role(s): Open PCR and Machine Testing
Name: Khalid Al-QahtaniRole(s): Research and Development
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.

Image: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

Image:WithoutDNA.jpg

  • Image J analysis of sample without DNA

Image: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
012352412.4461537377499658
01140818.257941977269357
015117618.6762823290710551
0.2515873476.60612160018787735
0.2513455247.0076324891326289
0.2513387148.1216442047428723
0.516693256.9919513592617318
0.514719432.2214742804394599
0.514638630.2384426407342671
111800881.26295896231098336
118321258.82810777923745412
1247655123.41530564391845749
2197697114.55822647709768839
2183426119.70821957473544822
2277605124.475345550211129608
5215236177.60238226246896635
5256884174.24844622241794800


Fitting a Straight Line


Image: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.


Image:Finalresults.jpg

  • Final Concentration Results
Personal tools