BME100 f2014:Group31 L5

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OUR TEAM

Name: Jimmy Xu
Name: Andrew Liu
Name: Andy Chang
Name: Charles Bolton
Name: Afshin Isadvesta
Name: Michael Chatarachanwong


LAB 5 WRITE-UP

Procedure

Smart Phone Camera Settings

  • Type of Smartphone: iPhone 5s
    • Flash:INACTIVE
    • ISO setting:800 OR HIGHER
    • White Balance: AUTO
    • Exposure: HIGHEST SETTING
    • Saturation: HIGHEST SETTING
    • Contrast: LOWEST SETTING


Calibration

The camera should be placed in a cradle perpendicular to the slide. The height of the camera should be adjusted so that it takes in the drop from the side. This can be altered using trays. After the correct height is found, the camera should be moved at least 4 cm away from the drop, so long as the image is not blurry.

  • Distance between the smart phone cradle and drop = 4 centimeters


Solutions Used for Calibration

2x Calf Thymus DNA Solution (μg/mL) Volume of 2x Solution (μL) Volume of SYBR Green I Dye Solution (μL) Final DNA Concentration (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. Pipette 80 μL of SYBR GREEN I dye in the center of the first two rows of the slide.
  2. Add 80 μL of 2x calf thymus solution on top of the SYBR GREEN I drop.
  3. Move the slide so that the 160 μL drop is directly aligned with the blue LED light.
  4. Take three images using the camera phone.
  5. Carefully remove the slide from the box.
  6. Using the pipette, suction the 160 μL of solution off the slide.
  7. Repeat steps 1-6 for each concentration of 2x calf thymus DNA solution, each time using a new pipette tip.


Data Analysis

Representative Images of Negative and Positive Samples

Positive Sample

Positive Sample.png

Negative Sample

Negative Sample.png

Image J Values for All Calibrator Samples


Final Concentration Area Mean Pixel Value RawIntDen Drop Raw IntDen Background Drop-Background
0 251912 112 28305047 850,368 27454679
0 217052 106.483 23112295 747350 22364945
0 223496 105.927 23674284 872669 22801615
0.125 134488 137.665 18514333 703726 17810607
0.125 133788 138.793 18568868 496871 18071997
0.125 122024 145.242 17722978 484699 17238279
0.25 280864 150.052 42144254 960204 41184050
0.25 278700 149.589 41690427 1092788 40597639
0.25 275672 149.821 41301404 1149072 40152332
0.5 416368 150.406 62624045 2813020 59811025
0.5 421316 148.636 62622846 2139682 60483164
0.5 423952 148.152 62809347 181147 62628200
1 58024 200.45 11630902 304312 11326590
1 58024 197.955 11486139 311127 11175012
1 57008 198.815 11334031 278956 11055075
2.5 85068 217.176 18474762 394075 18080687
2.5 94224 218.968 20632085 456385 20175700
2.5 84548 219.887 18591041 428213 18162828


Concentrations, means of background-subtracted values, and standard deviations

Final DNA Concentration RAWINTDEN Drop-Background 1 2 3 Mean STD
0 27454679 22364945 22801615 24207079.67 2820965.481
0.125 17810607 18071997 17238279 17706961 426413.2923
0.25 41184050 40597639 40152332 40644673.67 517464.6875
0.5 59811025 60483164 62628200 60974129.67 1471361.346
1 11326590 11175012 11055075 11185559 136064.4267
2.5 18080687 20175700 18162828 18806405 1186555.259

Calibration curve
Plot & Best-fit.jpg

  • Note* The distance between the phone and the drop were off for the images of the 2.5 and 1 µg/mL final DNA concentration in SYBR Green I solution, so the amount of light collected was affected, thus affecting our data and the best-fit line. We then just used the best-fit line of the example calibration curve provided in the lab workbook.

PCR Results Summary

Solutions Used for Measuring PCR Products:

PCR Product TUBE LABEL Volume of the DILUTED PCR Product solution (µL) Volume of the SYBR Green I Dye Solution (µL) Dilution 1 Dilution 2 Total Dilution (simplified fraction)
Plus 80 80 1/6 1/2 1/12
Minus 80 80 1/6 1/2 1/12
1--1 80 80 1/6 1/2 1/12
1--2 80 80 1/6 1/2 1/12
1--3 80 80 1/6 1/2 1/12
2--1 80 80 1/6 1/2 1/12
2--2 80 80 1/6 1/2 1/12
2--3 80 80 1/6 1/2 1/12
3--1 80 80 1/6 1/2 1/12
3--2 80 80 1/6 1/2 1/12
3--3 80 80 1/6 1/2 1/12


Raw and background-subtracted data for PCR

PCR Product Tube Label AREA Mean Pixel Value RAWINTDEN OF THE DROP RAWINTDEN OF THE BACKGROUND RAWINTDEN DROP - BACKGROUND
G31 P 1 148464 228.999 33998134 1402425 32595709
G31 P 2 134368 235.328 31620589 1119049 30501540
G31 P 3 145628 229.644 33442544 1238677 32203867
G31 N 1 210940 145.421 30675037 1188541 29486496
G31 N 2 198960 146.127 29073514 1162859 27910655
G31 N 3 196112 143.388 28120102 1171750 26948352
G31 1-1-1 231132 231.197 53437076 2201651 51235425
G31 1-1-2 213748 234.783 50184476 2020268 48164208
G31 1-1-3 227920 232.429 52975269 2085259 50890010
G31 1-2-1 209676 104.544 21920413 899646 21020767
G31 1-2-2 205842 111.89 23031746 859837 22171909
G31 1-2-3 204976 107.189 21971165 921155 21050010
G31 1-3-1 208214 235.522 49039025 895793 48143232
G31 1-3-2 195436 239.059 46720804 846868 45873936
G31 1-3-3 218512 227.497 49710931 938657 48772274
G31 2-1-1 140865 96.839 13641276 590821 13050455
G31 2-1-2 153480 100.59 15433734 671332 14762402
G31 2-1-3 147788 99.089 14644152 653159 13990993
G31 2-2-1 219580 90.784 19934254 1099260 18834994
G31 2-2-2 224072 87.421 19588540 1025358 18563182
G31 2-2-3 223433 88.204 19708723 1227490 18481233
G31 2-3-1 198748 79.371 15774911 1047658 14727253
G31 2-3-2 217582 87.12 18955755 1167237 17788518
G31 2-3-3 235280 85.438 20101761 1090183 19011578


Means of background-subtracted values for PCR

PCR Product Tube Label RAWINTDEN DROP - BACKGROUND ' ' '
1 2 3 MEAN
G31 P 32595709 30501540 32203867 31767038.67
G31 N 29486496 27910655 26948352 28115167.67
G31 1-1 51235425 48164208 50890010 50096547.67
G31 1-2 21020767 22171909 21050010 21414228.67
G31 1-3 48143232 45873936 48772274 47596480.67
G31 2-1 13050455 14762402 13990993 13934616.67
G31 2-2 18834994 18563182 18481233 18626469.67
G31 2-3 14727253 17788518 19011578 17175783


PCR Product Tube Label MEAN RAWINTDEN DROP - BACKGROUND PCR Product Concentration (µg /mL) Initial PCR Product Concentration (µg/mL)
G31 P 31767038.67 6.301311533 0.525109294
G31 N 28115167.67 5.570937333 0.464244778
G31 1-1 50096547.67 9.967213333 0.830601111
G31 1-2 21414228.67 4.230749533 0.352562461
G31 1-3 47596480.67 9.467199933 0.788933328
G31 2-1 13934616.67 2.734827133 0.227902261
G31 2-2 18626469.67 3.673197733 0.306099811
G31 2-3 17175783 3.3830604 0.2819217


  • Our positive control PCR result was 0.53 μg/mL
  • Our negative control PCR result was 0.46 μg/mL

Observed results

  • Patient 96107: The concentration of this patient's sample was an average of 0.66 μg/mL. When a drop of the sample was added into the SYBR Green I, it turned a shade of light green.
  • Patient 74312: The concentration of this patient's sample was an average of 0.27 μg/mL. When a drop of the sample was added into the SYBR Green I, it seemed to have no effect as no visible color change occurred.

Conclusions

  • Patient 96107: The patient's sample was closer to the positive control value. This means that the patient does have the disease because the genetic sequences being tested is similar to the positive control when being amplified.
  • Patient 74312: The patient's sample was close to the negative control value. This means that the patient does not have the disease because the genetic sequence being tested is similar to the negative control when being amplified.




SNP Information & Primer Design

Background: About the Disease SNP

SNP, or single nucleotide polymorphisms, are point mutations in the genetic sequence in which a single nucleotide, or one base unit within DNA, is altered. These can be silent (have no visible effect) or cause something as deadly as cancer. The gene in question is the KCNE2 gene, which is associated with Long QT syndrome. Located on the 21st human chromosome, this pathogenic gene is caused by a SNP. The point mutation is a simple switch of a thymine nucleotide to the cytosine nucleotide. The KCNE2 gene regulates potassium voltage gated channels, and can affect everything from neurotransmitter release to muscle construction to heart rate.

Primer Design and Testing

To design primer pairs for PCR, the non-disease forward primer contains 20 bases and ends with the aforementioned disease codon. The reverse non-disease primer also contains 20 bases, and is found 200 base pairs to the right on the bottom strand. To create disease primer pairs, the single nucleotide polymorphism is applied to the appropriate nucleotide in the forward primer; in this case, it is the thymine nucleotide. The reverse primer does not change when diseased.

These are screenshots of the web search results through the UCSC In-Silico PCR site.

This is the non-disease forward and reverse primer search.

1.png

This is the disease forward primer with the non-disease reverse primer search.

2.png