BME100 s2014:W Group6 L5

From OpenWetWare

Jump to: navigation, search
BME 100 Spring 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

Elizabeth BurkettRose Among the Thorns
Elizabeth Burkett
Rose Among the Thorns
Matthew WarrenThe Handy Man
Matthew Warren
The Handy Man
Samuel McCollumMan in Charge
Samuel McCollum
Man in Charge
Frederick SebastianWetWare Master
Frederick Sebastian
WetWare Master


LAB 5 WRITE-UP

Background Information

SYBR Green Dye
This is a solution of an asymmetrical dye used as a nucleic acid stain in molecular biology. It binds to the DNA and the new DNA strands emit a green light. It is useful for DNA amplification because it emits green light when there is a large amount of DNA in the solution.


Single-Drop Fluorimeter
The flourimeter has a box shape and is black in color. There is a place for a slide to be inserted on the top. A blue light is emitted across the slide to illuminate the sample.


How the Fluorescence Technique Works
The fluorescence technique works by using color to determine the presence of a disease. A clear sample will show that there is a negative test for the disease. On the other hand, a green sample proves that there is a disease present.



Procedure

Smart Phone Camera Settings

  • Type of Smartphone: iPhone 5
    • Flash: Off
    • ISO setting: Auto
    • White Balance: Auto
    • Exposure: Auto
    • Saturation: Auto
    • Contrast: Auto


Calibration

In order to calibrate the system, place the iPhone 5 in the cradle. Ensure that the camera is almost perfectly aligned with the slide with a sample drop that is exposed to the blue fluorimeter. The fluorimeter may need to be raised so that it is close to level with the camera. This can be done using small notebooks or small plastic plates that were provided in lab. Place a box over the system, making sure that no light enters when the pictures are being taken. Make sure the camera has a timer so that the box can be closed completely before the photo is taken. This process is repeated with different sample concentrations. ImageJ is the nused to analyze these images of different samples, and the RAWINTDEN values were then recorded. These values were used to make a calibration curve, of RAWINTDEN versus half-thymus DNA concentration.

  • Distance between the smart phone cradle and drop = 9.4 cm

Solutions Used for Calibration

Image:Calibration samplegrp8.jpg


Placing Samples onto the Fluorimeter

  1. Step 1: Insert the slide into the flourimeter and align it so that the front two dots on the slide are under the blue flourimeter.
  2. Step 2: Set the Micropipette to 80μL and pipette the SYBR Green dye onto the slide, change tips, and pipette the same amount of the patient DNA sample onto the same spot on the slide.
  3. Step 3: Start the timer on the camera and close the box completely to take the picture.
  4. Step 4: Set the Micropipette to 160μL and pipette all of the sample solution off of the slide, dispose of the tip, and move the slide to a new position for the next sample.


Data Analysis

Representative Images of Samples

[Instructions: Show an IMAGE where you drew a circle around the droplet with the freehand tool for a sample with no DNA]

Screen_Shot_2014-04-16_at_6.35.05_AM.png

[Instructions: Show an IMAGE where you drew a circle around the droplet with the freehand tool for a sample with DNA (positive signal)]

Screen_Shot_2014-04-16_at_6.34.53_AM.png

Image J Values for All Samples

[Instructions: See worksheet page 8. To save time on typing a new Wiki table from scratch, use THIS TOOL to auto-generate a Wiki table: Excel-to-Wiki Converter. Copy the headers and values from the Excel spreadsheet you made, paste them into the form field, click submit, copy the Wiki code that the tool generated, and replace TABLE GOES HERE (below) with your auto-generated code.]


Image:Intden_valuesgrp8.jpg


Fitting a Straight Line

[Instructions: Place an IMAGE of your Excel plot with a line of best fit here. See worksheet page 9] Calibrationcurveyolo.jpg.png

Report 5 Addendum

PCR Results Summary

Instructor's summary: You completed 8 PCR reactions in a previous lab. You used the SYBR Green I staining and imaging technique to measure the amount of amplified DNA in each PCR reaction. You used a standard curve (based on known concentrations of calf thymus DNA) to convert INTDEN values into DNA concentration. Your positive control and negative control samples should be used as threshold values for determining whether an unknown (patient) sample is truly positive or negative.

Important Note: The following concentrations are negative. While it is impossible for such measurements to be correct, they may still function as indicators of the presence of the disease by comparison to the negative and positive control.

positive control PCR result was -1.18094 μg/mL

negative control PCR result was -4.52187 μg/mL

Write-in each patient ID and give both a qualitative (what the images looked like) and a quantitative description (μg/mL) of what you observed

Patient 1 (ID: 67618): Average concentration calculated: -4.51273μg/mL. Qualitative analysis: There is no or minimum fluorescence in each image of Patient 1's DNA.

Patient 2 (ID: 78379): Average concentration calculated: -4.09297μg/mL. Qualitative analysis: There is no or minimum fluorescence in each image of Patient 2's DNA.

Compare each patient's results to the positive control value and the negative control value. Draw a final conclusion for each patient (positive or negative) and explain why you made that conclusion.

Patient 1: The patient's average concentration of DNA is very close to the negative control. Additionally, the lack of observed fluorescence in this patient's DNA is markedly different from the positive control, which was visibly green. Therefore, Patient 1 is judged to be free of the disease (a negative result).

Patient 2 : The patient's average concentration of DNA is lower than that of the other patient's, but still far closer to the negative control than the positive control. Patient 2 also lacked visible fluorescence. Therefore, Patient 2 is also negative for the disease.

Personal tools