BME100 f2014:Group11 L5

From OpenWetWare

Jump to: navigation, search
BME 100 Fall 2014 Home
People
Lab Write-Up 1 | Lab Write-Up 2 | Lab Write-Up 3
Lab Write-Up 4 | Lab Write-Up 5 | Lab Write-Up 6
Course Logistics For Instructors
Photos
Wiki Editing Help
Image:BME494_Asu_logo.png


Contents

OUR TEAM

Name: Aliya Yano
Name: Aliya Yano
Name: Breanna Corrigan
Name: Breanna Corrigan
Name: Julian Lopez
Name: Julian Lopez
Name: Carlos Cabanes
Name: Carlos Cabanes
Name: Mohammed Almaimani
Name: Mohammed Almaimani


LAB 5 WRITE-UP

Procedure

Smart Phone Camera Settings

  • Type of Smartphone: iPhone5S
    • Flash: No Flash
    • ISO setting:800
    • White Balance: Auto
    • Exposure:Auto
    • Saturation:Auto
    • Contrast:Auto
    • Set phone to video mode then simply took three screenshots of to get our pictures


Calibration

  • Distance between the smart phone cradle and drop = 5cm


Solutions Used for Calibration

Initial Concentration of n2X 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)
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. Choose a camera to use, set it to the specifications described in the workbook and place it in the stand.
  2. Set up the Flourimeter.
  3. Place slide in the flourimeter with the smooth side down and the rough side up.
  4. Place one, 80 micro-mL drop of SYBR Green on the slide, between two of the dots on the slide.
  5. Place one, 80 micro-mL drop of a calf thalmus solution on the slide so it combines with the SYBR Green.
  6. Adjust the slide so the light shines through the drops.
  7. Adjust and record camera distance.
  8. Start camera video and close the fluorimeter.
  9. Wait a few seconds and remove camera, take screenshots on the video at different times and send them to be analyzed.
  10. Discard the droplets and slide.
  11. Repeat.


Data Analysis

Representative Images of Negative and Positive Samples


Positive
Positive Sample


Negative
Negative Sample


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
2.57259227.453199287401992874
2.58184024.854203406062034054
2.56912435.062242362802423628
110074832.7373298170693298101
19032036.084325907363259067
19039236.4033290496673290429
0.5357818.65866760160565155
0.55208815.4518047921540803252
0.56245213.068156282564813064
0.2511307616.741189295499351883019
0.256830826.692182327896041813674
0.256012829.933179982284921791330
0.1257101627.5281954937131771941760
0.1255248035.86188191432041878710
0.125731447314427.1934377-4349.807
06716428.526191593548631911072
07847221.653169917731261696051
07329622.284163334113431631998



Final DNA Concentration in SYBR Green I Solution (µg/mL) 1 2 3 Average Standard Deviation
2.53298101325906732904293282532.33320680.45689
11992874203405424236282150185.333237701.7397
0.5188301918136741791330182934147810.13854
0.251911072169605116319981746373.667146184.3099
0.125194176018787104349.8071274939.9361100814.826
065155803252813064560490.3333429001.0352


Calibration curve

calibration
Image J Values for DNA Samples

PCR Product Tube Label Area Mean Pixel Value RAWINTDEN of the drop RAWINTDEN of the Background RAWINTDEN DROP - BACKGROUND
P6693628.8471930873483189604
P4708037.904178453533961781139
P5834032.315188523082971876933
N3647234.757126765201267652
N5936022.418133070301330703
N4870027.631345571161345555
1.15396425.7151387673161387657
1.15233235.469185616865751849593
1.15635632.198181454415741812970
1.23961634.895138238901382389
1.25153627.49141673201416732
1.26975624.391145748601457486
1.35040436.57618435552731843282
1.35065637.629190611666161899500
1.3628726.1161641941234162960
2.16522427.2751778976701778906
2.14335638.869168520124801682721
2.1452631.611143071212142859
2.26588037.459246778931492464640
2.26506435.55623133906832312707
2.2583835.754208730837207893
2.33708037.087137518725081372679
2.34290433.525143837432561435118
2.33625636.62413278254001327425



PCR Product Tube Label 1 2 3 Average Standard Deviation
P189604178113918769331282558.667947737.5956
N1267652133070313455551314636.66741361.99527
1.11387657184959318129701683406.667256780.4705
1.2138238914167321457486141886937594.08104
1.3184328218995001629601301914805689.0535
2.1177890616827211428591201495.333918066.4754
2.2246464023127072078931661746.6671261363.854
2.31372679143511813274251378407.33354074.54035



Calculating Concentration of PCR Product

PCR Product Tube Label MEAN RAWINTDEN DROP - BACKGROUND PCR PRODUCT CONCETRATION INITIAL PCR PRODUCT CONCENTRATION
P1282558.6670.3130864773.75703772
N1314636.6670.3486302034.183562439
1.11683406.6670.7572423389.086908057
1.214188690.4641238615.569486335
1.313019140.3345329724.014395664
2.11201495.3330.2232650122.679180139
2.21661746.6670.7332421798.798906142
2.31378407.3330.4192906915.031488298

PCR Results Summary

  • Our positive control PCR result was 3.757 μg/mL
  • Our negative control PCR result was 4.183 μg/mL

Observed results

  • Patient 81166 : All images taken of patient one's DNA samples seemed to be negative. None of them glowed green. All of this patients concentration results were above the negative control.
  • Patient 54283 : All images taken of patient two' DNA samples appeared to be negative as well as none of them glowed green like the positive sample did. However, not all of the values of concentration found for patient two were above the negative control.

Conclusions

  • Patient 81166 : Based on the images alone, this patient tested negative.The data calculated shows all concentrations to be above both the negative and positive controls which suggests the sample to be negative as the negative concentration was found to be higher than the positive. The final conclusion is this patient is negative.
  • Patient 54283 : Based on the images alone, this patient tested negative. The data calculated shows two of the three concentrations to be above both the negative and positive control and one below. This still impliess that this patient is negative.

Examining only the data collected, it is hard to determine a result as all the data found using Image J is relatively similar. The positive and negative control concentrations were very near to each other with the negative control having a higher concentration than the positive control. The data collected for the patients ranged both above and below the controls.This suggests an error was made and further testing would be needed to get a definitive result.




SNP Information & Primer Design

Background: About the Disease SNP A nucleotide is a subunit of a nucleic acid and a polymorphism is when two or more clearly different phenotypes exist within the same population. Therefore, a SNP, or single nucleotide polymorphism, is simply a DNA sequence variation where a single nucleotide is different when compared to others of the population. The variation rs1699154 is found in Homo Sapiens a.k.a humans. It is located on the chromosome 21:34370656. This SNP is pathogenic and associated with the KCNE2 gene-- which stands for potassium voltage-gated channel-- and the cardiac disease Long QT Syndrome.

Primer Design and Testing The KCNE2 gene stands for potassium voltage-gated channel Isk-related family, member 2. The non-disease allele is TTC but the disease-associated allele contains the sequence is CTC. The numerical location of this SNP is 34370656. The numerical position needed for the non-disease reverse primer is exactly 200 bases to the right of the SNP at 34370856. the non-disease forward primer is 5'-catggtgatgattggaatgt, the reverse primer sequence is 5'-cccttatcagggggacattt. Similarly, the disease forward primer is 5'-catggtgatgattggaatgc and the disease reverse primer is cccttatcagggggacattt.

calibration calibration

Personal tools