BME100 s2014:T Group9 L5

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Contents

OUR TEAM

Name: Cassaundra Kinnear
Name: Cassaundra Kinnear
Name: Katherine Underwood
Name: Katherine Underwood
Name: Kyle Queen
Name: Kyle Queen
Name: Mohammed Binsheliail
Name: Mohammed Binsheliail


LAB 5 WRITE-UP

Background Information

SYBR Green Dye

SYBR green dye is used as a nucleic acid stain and binds to DNA. The result is a DNA-dye-complex that absorbs blue light and emits green light. This dye can also bind to single-stranded DNA and RNA with a lower performance.


Single-Drop Fluorimeter

A single-drop fluorimeter is made up of a sturdy black cover used to block out the light. There is a phone cradle used o hold the camera while taking pictures. Inside the black cover there is a platform to hold the slides. There is also an LED light placed around the slides. This light emits a blue light used to see the SYBR green dye.


How the Fluorescence Technique Works

The blue LED light shines on the DNA-dye samples. The amount of DNA in each sample is measured based on the concentration of the green light being emitted from the sample.



Procedure

Smart Phone Camera Settings

  • Type of Smartphone: iPhone 4s
    • Flash: off
    • ISO setting: N/A
    • White Balance: N/A
    • Exposure: N/A
    • Saturation: N/A
    • Contrast: N/A


Calibration

The camera was set on a cradle eye level with the sample. We set the camera with a timer so there would be no other light than the LED light the green glow coming from the sample. We clicked the button and closed the flap of the outer black case, then the camera took the picture of the sample.

  • Distance between the smart phone cradle and drop = 8 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)
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. Place the fluorimeter into the box
  2. Place the camera in the cradle
  3. Adjust the height of the fluorimeter with the trays so the camera will see the drop at eye level
  4. Insert the slide into the fluorimeter
  5. Adjust the distance between the camera and the slide to be as close as possible without being blury
  6. Place an 80 microliters drop of SYBR Green in between the first two rows of the slide
  7. Add 80 microliters of the indicated DNA solution to the drop of SYBR Green
  8. Align the sample and the blue LED light
  9. Click the timer on the camera and close the lid for the picture
  10. Take one more picture, for a total of two pictures, without moving the camera



Data Analysis

Representative Images of Samples

Image:Droplet_(2).png

Image:Droplet_2_(2).png


Image J Values for All Samples

Concentration Area Mean IntDen RawIntDen RawIntDen Back
08982422.134198814219881424075684
10602425.339268650426865043520178
24442039.829973507097350704252614
0.2523262054.6512712759127127591470699
21309660.94412986996129869961545487
23040843.88510111560101115601460134
0.531608886.67827397838273978385561052
263648111.59229421095294210955410757
228141134.4730678127306781271187005
1275068121.33533375368333753681276411
285964116.94433441633334416331345189
99532113.98411345102113451021352381
2101468114.7391164236311642363921896
101884125.7411281098712810987957122
109756115.5512682322126823221114350
5108804173.9761892923718929237654847
109360120.3321315948013159480796070
106772111.9491195299911952999918322

Excel-to-Wiki Converter.


Tube A Tube B Tube C Tube D Tube E Tube F Tube G Tube H
230822141301555069909532515711018266146303611757997421359877
407856947735635282577575305511153155167802321826854121671092
410724950557475291947764447213454922171488502026057522388687


Fitting a Straight Line

Image:Graph_(2).png



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