BME100 s2015:Group8 12pmL5

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OUR TEAM

Name: Priscilla Hernandez
Name: Alwaleed Alsahafi
Name: Victoria Sanford
Name: Sarah Soaf
Name: Taylor Simone Woods


LAB 5 WRITE-UP

Procedure

Smart Phone Camera Settings

  • Type of Smartphone: Samsung Galaxy SIII
    • Flash: off
    • ISO setting: auto
    • White Balance: auto
    • Exposure: 0
    • Saturation: 0
    • Contrast: 0


Calibration

  • Distance between the smart phone cradle and drop = 10 cm


Solutions Used for Calibration

Initial Concentration of 2X Calf Thymus DNA Solution (μg/mL) Volume of the 2X DNA Solution (μL) Volume of the SYBR Green I Solution (μL) Final Concentration of 2X Calf Thymus DNA Solution (μg/mL)
0 80 80 0
0.25 80 80 0.125
0.5 80 80 0.25
1 80 80 0.5
2 80 80 1
5 80 80 2.5



Placing Samples onto the Fluorimeter

  1. Put on gloves. Then find the smooth side of the slide.
  2. Retrieve a fluorimeter from the TA and take it to your group's table.
  3. Put the slide from before in its place on the fluorimeter, making sure the smooth side is facing down.
  4. Open the camera app, turn on the timer for 3-5 seconds, and put it on the craddle.
  5. Make sure the camera has a good view of the slide by adjusting either the height of the fluorimeter or the camera.
  6. Using a micropipette, take 80 micro liters of SYBR Green I solution and carefully put it between the front two clear circles of the slide.
  7. Now put 80 micro liters of the sample/calibration over the SYBR Green I drop.
  8. Make sure the that the light is hitting the drop in the middle and goes through the other side by adjust the slide.
  9. The camera should be at closest 4 centimeters and focused. Record how far the camera is from the fluorimeter.
  10. Cover the fluorimeter with the lightbox with one of the flaps up.
  11. Double check that the camera is focused on the drop.
  12. Start the camera's timer and take the last flap down.
  13. Once the picture is taken, dispose of the 160 micro liter drop.
  14. Move on to the next position on the slide.
  15. Repeat all the steps for each sample.


Data Analysis

Representative Images of Negative and Positive Samples

Pic7.jpg Pic8.jpg


Image J Values for All Calibrator Samples


Sample Area Mean Min Max IntDen
H2O 122472 22.478 0 255 2752947
0.25 334592 33.476 0 255 11200689
0.5 193988 44.834 0 255 8697194
1 117636 55.702 0 255 6552507
2 189104 71.432 0 255 13506310
5 147412 73.233 0 255 10795378
Negative 98980 70.295 0 255 6957848
Positive 84628 73.603 0 255 6228875
Patient2A 73900 37.997 0 255 2807950
Patient2B 76620 41.83 0 255 3205014
Patient2C 184944 84.546 0 255 15636268
Patient1A 183223 61.598 0 255 11286081
Patient1B 107880 39.85 0 255 4299046
Patient1C 90812 65.6 0 255 5957275


Pic1.jpg Pic2.jpg Pic3.jpg Pic4.jpg Pic5.jpg Pic6.jpg Pic9.jpg Pic10.jpg Pic11.jpg Pic12.jpg Pic13.jpg Pic14.jpg

Calibration curve
Group 8 noon.jpg


PCR Results Summary

  • Our positive control PCR result was 80 μg/mL
  • Our negative control PCR result was 80 μg/mL

Observed results

  • Patient 1 (ID: 62525): The one sample with fluorescence seemed similar to the 2 μg/mL sample
  • Patient 2 (ID: 86787): There was a slight observance of SYBR Green which seemed to range from nothing to .5

Conclusions

  • Patient 1 (ID: 62525) : This patient possibly has SNP. The first two samples were cracked and the third appeared much more fluorescent so perhaps the amount of sample was not enough for detection when diluted.
  • Patient 2 (ID: 86787) : This patient does not have SNP.




SNP Information & Primer Design

Background: About the Disease SNP The Single Nucleotide Polymorphism (SNP) is a DNA sequence variation. One of the known SNPs is rs268 in the lipoprotein lipase (LPL) gene. rs268 is found in a species called Germline, which is a series of germ cells. This variation is located in chromosome 8: 19956018. It is considered as a pathogenic SNP and is associated with Coronary Heart disease.

Primer Design and Testing

After testing the Non-disease primer, both forward and backward, we got successful results assuming calculating it with 50mM salt and 50nM annealing oligo concentration. However, the disease primer testing was not successful where there was " no match found."


Non-disease reverse and forward primer: Non Disease.jpg Non-Disease temp.jpg


Disease reverse and forward primer:


Disease no match.jpg Disease temp.jpg