BME100 s2015:Group8 12pmL5

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Contents

OUR TEAM

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

Image: Pic7.jpg Image: Pic8.jpg


Image J Values for All Calibrator Samples


Sample Area Mean Min Max IntDen
H2O12247222.47802552752947
0.2533459233.476025511200689
0.519398844.83402558697194
111763655.70202556552507
218910471.432025513506310
514741273.233025510795378
Negative9898070.29502556957848
Positive8462873.60302556228875
Patient2A7390037.99702552807950
Patient2B7662041.8302553205014
Patient2C18494484.546025515636268
Patient1A18322361.598025511286081
Patient1B10788039.8502554299046
Patient1C9081265.602555957275


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

Calibration curve
Image: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: image:Non Disease.jpg image:Non-Disease_temp.jpg


Disease reverse and forward primer:


image:Disease_no_match.jpg image:Disease_temp.jpg

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