BME100 s2014:T Group4 L5

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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: Brian Kalen
Name: Michael Spina
Name: Daniel Munoz
Name: Parker Davis
Name: Rakan Aldrssary


LAB 5 WRITE-UP

Background Information

SYBR Green Dye

A photosenstive dye that reacts with certain DNA strands. The dye helps quantify how much DNA is present and therefore a sample with more DNA will appear more green. SYBR green stains the DNA strands in order to show how much DNA is in a sample when a beam of light is shot through the sample. SYBR green in high concentration is used in PCR similar to the lab performed. This helps us find out how much DNA is in a certain sample. When there is little to no DNA in the sample the dye will not stain as much and by consequence the sample will not shine with a green glow when light is shot through it. The SYBR green dye works best with double stranded DNA however, it has been shown to stain single stranded DNA and in some cases even RNA.

Single-Drop Fluorimeter

Unnamed1.png

This single drop fluorimeter is a box that covers a black stand that can hold a bubble of solution.
A slide is placed on the fluorimeter and the DNA sample is placed in drop form on the slide. The slide has a grid of dots, and the sample solution is placed in between the dots on the slide in order to obtain the clearest measurments. The fluorimeter has a light switch on the side, and when the switch is on a beam of fluorescent light is shot across the device. When bubble of solution is placed on the slide the beam of light penetrates the solution and reflects certain contents of the solution. The box is used to cover the sample in order to maximize the ability to see how much green stained DNA was present.


How the Fluorescence Technique Works


The DNA sample is placed on a slide and then shifted into the box so the sample is in complete darkness.
The sample is sent into darkness so that no other light will effect how much green dye is reflected. Fluorescent light is then shot through the bubble in order to reflect how much DNA is present through how much green dye is displayed. Based on how much Green in this case is shown will show us how much DNA is present in the sample. The light pierces the bubble of solution, and the stained DNA strands are reflected as a green color due to the SYBR green dye.



Procedure

Smart Phone Camera Settings

  • Type of Smartphone: Galaxy S 3 from Samsung
    • Flash: None
    • ISO setting: 800
    • White Balance: auto
    • Exposure: +2.0
    • Saturation: auto
    • Contrast: auto



Calibration


The phone was placed in a holding tray so it could stand up on its own. Then the phone was set so that it had a timer and would take a picture 5 seconds after the camera was pushed.
This gave us the time to close the box and allow the sample to be in complete darkness. This yielded the highest quality pictures for measurement. Unnamed2.png


  • Distance between the smart phone cradle and drop = 7cm.


Solutions Used for Calibration

C+ ID: 31526 ID: 31526 ID: 31526
C+ P11 P12 P13
C- ID: 36055 ID: 36055 ID: 36055
C- P21 P22 P23



1. Placing Samples onto the Fluorimeter

2. Placed 80 microliters of SYBR Green in between the two middle circles of the slide.


3. Placed 80 microliters of DNA solution on top of the Green dye bubble.


4. Then the light was turned on from the side of the fluorimeter.


5. The camera timers was started and the fluorimeter was pushed to the back of the dark box.


6. The back lid of the box was shut to ensure the sample was in complete darkness.


7. Wait five seconds for the camera timer to expire, and then take the sample out and save the picture for analysis.


Data Analysis

Representative Images of Samples

Negative Control

Negativecircled.png

Positive Control

Greencircled.png


Image J Values for All Samples

{|

Final DNA Concentration RAWINTDEN of drop RAWINTDEN of background RAWINTDEN drop - RAWINTDEN background
2.5 10001162 72782 9928380
2.5 6726675 40465 6686210
2.5 6571887 40455 6531432
1 4366858 38133 4328725
1 4393627 37731 4355896
1 4416778 37069 4379709
0.5 3171170 40433 3130737
0.5 3187197 39794 3147403
0.5 3113140 43137 3070003
0.25 1751859 37579 1714280
0.25 2062352 43553 2018799
0.25 1979956 42452 1937504
0.125 1315149 38227 1276922
0.125 1271349 40257 1231092
0.125 1355536 43030 1312506
0 478359 40699 437660
0 557450 39977 517473
0 448736 43522 405214


Fitting a Straight Line

INTDEN.png



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.

Your positive control PCR result was 0.241 μg/mL
Your negative control PCR result was -0.00028 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 31526 : Sample was nonreactive with SYBR Green 1 and showed no green coloration under fluorescent light
Patient 36055 : Sample was nonreactive with SYBR Green 1 and showed no green coloration under fluorescent light

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 31526 : This patient was negative. The corrected PCR Product Concentration strongly resembled the negative control value
Patient 36055 : This patient was negative. The orrected PCR Product Concentration had a value much closer to the negative control than the positive.


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