BME100 s2015:Group16 12pmL5

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Lab Write-Up 1 | Lab Write-Up 2 | Lab Write-Up 3
Lab Write-Up 4 | Lab Write-Up 5 | Lab Write-Up 6
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OUR TEAM

Name: Hanna Monroe
Name: Natkanes Pakjirasirikul
Name: Trevaun Walker
Name: Reaghan Fletcher
Name:Thomas Murphy


LAB 5 WRITE-UP

Procedure

Smart Phone Camera Settings

  • Type of Smartphone: ZTE N9180
    • Flash: No Flash
    • ISO setting:400
    • White Balance:Auto
    • Exposure:Highest
    • Saturation:Highest
    • Contrast:Lowest


Calibration

G16calibration.png

  • 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 (μL)
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. Turn on Blue LED Excitation light
  2. Insert a slide into the Fluorimeter
  3. Place camera on the craddle and as close to as possible without getting a blurry picture
  4. Adjust height of fluorimeter so that picture from camera takes a picture of the drop directly from the side
  5. Place an 80 microliter drop of SYBR Green I on the slide so that it looks like a beach ball
  6. Add an 80 microliter drop of the DNA solution to the already placed solution
  7. Align the slide so that the blue LED is focused on the drop
  8. Take the a picture with the timer set and lower the lid of the lightbox
  9. Take a total of 3 pictures then remove the solution from the slide
  10. Move the slide to the next position and repeat the process with the different solutions


Data Analysis

Representative Images of Negative and Positive Samples

Positve:

Positvejpg.jpg


Negative:

Negativejpg.jpg


Image J Values for All Calibrator Samples


Final DNA concentration in SYBR Green I solution (μg/mL) Area Mean Pixel Value RawIntDen of the drop RawIntDen of the background RawIntDen drop - background
2.5 139380 108.933 15183035 37084 15145951
2.5 142636 109.163 15570617 37393 15533224
2.5 147752 107.308 15854933 49037 15805896
1 106708 83.381 8897448 17939 8879509
1 115956 80.25 9305435 21998 9283437
1 113112 90.715 10260974 34066 10226908
0.5 135592 62.432 8465278 64454 8400824
0.5 118410 58.929 6977741 64902 6912839
0.5 115368 58.331 6729582 46971 6682611
0.25 139968 40.608 5683780 59072 5624708
0.25 127236 40.933 5208100 54709 5153391
0.25 133808 42.526 5690375 51402 5638973
0.125 131884 38.479 5074747 49047 5025700
0.125 124424 34.43 4283904 43969 4239935
0.125 132332 37.836 5006942 54115 4952827
0 137400 21.711 2983023 52412 2930611
0 124400 18.681 2323902 45159 2278743
0 114528 19.95 2284888 39148 2245740


Final DNA concentration in SYBR Green I solution (μg/mL) RawIntDen drop - background ' ' ' Standard Deviation
1 2 3 mean
2.5 15145951 15533224 15805896 46485071 331626.7478
1 8879509 9283437 10226908 28389854 691469.3802
0.5 8400824 6912839 6682611 21996274 932680.7426
0.25 5624708 5153391 5638973 16417072 276325.015
0.125 5025700 4239935 4952827 14218462 434156.6754
0 2930611 2278743 2245740 7455094 386235.9758



Calibration curve

G16calcurve.png


PCR Results Summary

  • Our positive control PCR result was .11603 μg/mL
  • Our negative control PCR result was .0575 μg/mL

Observed results

  • Patient 47360 : Average initial concentration of .114425, the bubble observed was clearer than the positive control, however much darker than the negative
  • Patient 31303 : Average initial concentration of .15928, the bubble was much darker the negative control and the patient 1, much closer to the color of the positive control.

Conclusions

  • Patient POS : 2 out of three of the runs ran closer in similarity to the positive control
  • Patient POS : All three of the runs were closer to the positive test




SNP Information & Primer Design

Background: About the Disease SNP

An SNP is a single nucleotide polymorphism(SNP). An SNP occurs when a nucleotide pair, the monomers that create DNA, is altered changing the gene it is contained in creating a polymorphism, a genetic variation. These SNPs occur naturally in biological genomes and through natural selection these mutations are controlled. When an SNP occurs it creates a new allele, a variation in the gene, in a population that if not detrimental will be passed on to the next generation. It is usual for there to be only two different alleles for a specific gene which is the case for the SNP being studied. The disease SNP being studied is rs268. This SNP occurs in Homo Sapiens on the eighth chromosome. The clinical significance of this SNP is that it is pathogenic and it is associated with the LPL gene, the lipoprotein lipase. This gene is involved with processes such as apolipoprotein binding, chylomicron remodeling, and anchoring components of the membrane. This disease is responsible for metabolic syndrome that increase blood pressure, cholesterol levels, excess fat, and blood sugar levels which increases a person risk for stroke, diabetes, and heart disease. The normal allele of this gene is AAT but when the disease is present the mutation creates an allele of AGT.


Primer Design and Testing

The primers worked as expected producing one positive test and one negative test. In the positive test the diseased primer was shown to work. In the negative test the non-disease primer was shown to work. The primers worked by bonding to the DNA and allowing replication to occur if they matched properly. By getting replicated DNA it was confirmed that the primers worked because they were attached. The primers were different at two different points in the forward direction. The non-disease primer would bond allowing replication if the allele was negative for the disease because it ended in an 'A' nucleotide which the gene has if it does not have the disease. The disease primer bonded and allowed replication to occur if the diseased allele was because it ended in a 'G' which the gene would have in at that location if the disease was present. The two nucleotide primers can be seen below.


Non-Disease Primer Primer Valid.jpg


Disease Primer Primer Invalid.jpg