BME100 s2015:Group10 12pmL5

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Contents

OUR TEAM

Name: Joseline Valenzuela
Name: Joseline Valenzuela
Name: Itai Kreisler
Name: Itai Kreisler
Name: Clayton Nunn
Name: Clayton Nunn
Name: Isaac Clouse
Name: Isaac Clouse


LAB 5 WRITE-UP

Procedure

Smart Phone Camera Settings

  • Type of Smartphone: Galaxy S5
    • Flash: Off
    • ISO setting: 800
    • White Balance: Auto
    • Exposure: +2.0
    • Saturation: Auto
    • Contrast: Auto


Calibration


  1. Turn on the Blue LED light on the fluorimeter.
  2. Adjust the camera settings on the smartphone if possible.
  3. Turn on camera.
  4. Place the smartphone on the cradle and adjust its distance from the fluorimeter and record it.
  5. Use plastic trays to adjust the height of the fluorimeter so the smartphone take a picture of the side of the slide.
  6. Be careful to keep the position of the smartphone consistent, otherwise the results could be affected.


Image:set_up_group_10.jpg

  • Distance between the smart phone cradle and drop = 6 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 I Dye solution (µL) Final DNA concentration in SYBR Green I solution (µg/mL)
580*80*2.5
280*80*1
180*80*0.5
0.580*80*0.25
0.2580*80*0.125
080*80*0


'Placing Samples onto the Fluorimeter

  1. Use a micro pipettor to transfer 80 microliters of SYBR Green onto the rough side of the slide. Make sure to apply the solution between two circles in order to form a spherical shape.
  2. Replace micropipettor tip.
  3. Add 80 microliters of one of the calf thymus solutions to the SYBR green drop.
  4. Move the slide so the solution is aligned with the blue LED.
  5. Take a picture by using the timer tool on the camera.
  6. Cover the fluorimeter from any light while taking the picture by placing a lid on the box.
  7. Take 3 pictures of every sample of the drop while the camera is focused.
  8. Once finished taking the picture, carefully remove the lid on the box. Try not to change the position of the smartphone.
  9. Remove the 160 microliters of solution using a micropipettor.
  10. Replace micropipettor tip.
  11. Repeat steps 7-14 for all of the concentrations of calf thymus DNA.


Data Analysis

Representative Images of Negative and Positive Samples

  • Negative Sample:

Image:NEG_GROUP_10.jpg

  • Positive Sample:

Image:POS_GROUP_10.jpg


Image J Values for All Calibrator Samples

Final DNA concentration in SYBR Green I solution (ug/mL) AREA Mean Pixel Value Rawintden of the drop Rawdinten of the background Rawdinten drop - background
513499784.5241145470715369011301017
513621286.7041181010315879211651311
513661385.681170506913560011569469
115466461.98495867271904229396305
116327665.5841070822321073710497486
116256164.8131053608423432310301761
0.515952640.80665096551792136330442
0.515439042.28165277151896586338057
0.514750842.30762406651980546042611
0.2513245630.60940543511558043898547
0.2513362029.46439369911235733813418
0.2513262831.96842399091402054099704
215169589.1071351704821707113299977
214946591.9441374240824430013498108
215437891.8111417367023294813940722
012570012.04815143801423041372076
014086511.9216790601653241513736
014459612.45718012591586801642579


Calibration curve
Image:g10calicurve.jpg

***Note: Our camera was not sensitive enough to capture the high concentrations of SYBR green. As a result we elected to exclude the last data point in order to obtain a more accurate trend line.***

PCR Results Summary

PCR Product TUBE LABEL RAWINTDEN DROP- BACKGROUND PCR Product Concentration (ug/mL) Initial PCR Product Concentration (ug/mL)
10+180023532.5003921671.5002353
10 1-1180463962.5077326671.5046396
10 1-2194469372.7411561671.6446937
10 1-3207155342.9525891.7715534
10 -103484751.2247458330.7348475
10 2-192261101.0376850.622611
10 2-277337640.7889606670.4733764
10 2-395267901.0877983330.652679
  • Our positive control PCR result was 1.500 μg/mL
  • Our negative control PCR result was .7348 μg/mL

Observed results

  • Patient 67713 : This patient's samples were fluorescent (green). The average initial PCR product concentration was 1.64(μg/mL), which is above the initial PCR concentration of the positive control of 1.500 (μg/mL), like all samples of this patient.
  • Patient 89702 :The images for patient two looks darker than our negative control for these samples. Also, our values for the μg/mL were .62, .47, .65 for each sample of this patient. Since all the values were under our negative control, it is correct to assume that this patient did not suffer from the SNP.

Conclusions

  • Patient 67713 : Based on the similarities of the initial PCR concentrations of the positive control and patient 67713, it is safe to assume that the patient tested positive for SNP. As mentioned above, the high fluorescence and the values of PCR concentration of the positive control closely matched this patient.
  • Patient 89702 :As mentioned above, each concentration calculated from the calibration curve yielded a concentration lower than that of our negative control. Thus we ascertained that this patient did not have the SNP, based on the low numbers in the previous section.




SNP Information & Primer Design

Background: About the Disease SNP

SNP stands for "single nucleotide polymorphism". This occurs when there is a discontinuous genetic variation on a single nucleotide, resulting in different types of individuals. This variation is found in Homo sapiens on chromosome 8. Variations in SNP can cause Hyperlipidemia. This SNP is associated with genes LIPD and HDLCQ11. According to summaries of research on PubMed, SNP is linked to coronary heart disease. It is believed to affect cholesterol levels, which in turn can lead to stroke and other related illnesses.


Primer Design and Testing


Image:RESULTS_GROUP_10_2.jpg Image:RESULTS_FAIL_GROUP_10_2.jpg


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