BME100 s2014:T Group12 L5

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Contents

OUR TEAM

Name: Casey Weigel
Name: Casey Weigel
Name: Spencer Brown
Name: Spencer Brown
Name: Kevin Haas
Name: Kevin Haas
Name: Mariama Salifu
Name: Mariama Salifu
Name: Davis Matthews
Name: Davis Matthews

LAB 5 WRITE-UP

Background Information

SYBR Green Dye
SYBR green dye binds to double stranded DNA. The dye absords blue light and emits green light.


Single-Drop Fluorimeter
A fluorometer or fluorimeter is a device used to measure parameters of fluorescence: its intensity and wavelength distribution of emission spectrum after excitation by a certain spectrum of light.
Image:BME.jpg

How the Fluorescence Technique Works
Once you turn the fluorimeter on and place it on the table you put the glass slide on smooth side down. Next set up the camera in the cradle so you are looking level at the slide, you might need to adjust the height. Turn your photo app on to be ready to take pictures. Next place 80 uL drop of SYBR Green I solution on first two clear circles in the middle of the slide. After that place 80 uL drop of DNA sample on top of the SYBR green I solution. Then adjust slide so that the light illuminates the center of the drop and the drop focuses the light on the other side. Finally find the distance between the camera and the fluorimeter to get a clear and well focused picture. Then record the distance and the pictures taken of each sample.



Procedure

Smart Phone Camera Settings

  • Type of Smartphone: Iphone 4
    • Flash: Off
    • ISO setting: >800
    • White Balance: Auto
    • Exposure: High
    • Saturation: High
    • Contrast: Low



Calibration


Set the cradle so that when the phone is placed, it will be perpendicular to the slide. Make sure that the camera can only see a small portion of the slide; the drop is the focus here. If necessary, raise the fluorimeter so that the slide is mostly unseen by the camera. Record the distance between the camera and the drop.

  • Distance between the smart phone cradle and drop = approximately 4 cm


Solutions Used for Calibration

Initial Concentration of 2X Calf Thymus DNA solution (micrograms/mL) Volume of the 2X DNA solution (uL) Volume of the SYBR GREEN I Dye solution (uL) Final DNA concentration in SYBR Green I solution (ug/mL)
5 80 80 2.5
2 80 80 1
1 80 80 .5
.5 80 80 .25
.25 80 80 .125
0 80 80 0


Placing Samples onto the Fluorimeter

  1. Place 80 microliters of SYBR green dye on the slide, on either end but between two of the circles. Then add 80 microliters of the DNA containing solution.
  2. Move the slide so that the drop is being hit by the blue LED light.
  3. Cover the system and take a delayed photo to allow time to close off most external light. Do not move the camera for any reason.
  4. Remove the drop that is on the slide and move to the next two circles on the slide. Return to step 1.


Data Analysis

Representative Images of Samples
Sample Without DNA

Sample With DNA


PCR Results Summary

PCR INTDENS Values and Concentrations Using Fluorescence

PCR Product TUBE LABEL AVERAGE INTDENS VALUE PCR Product Concentration, µg /mL Corrected PCR Product Concentration, µg /mL
PC 14842638.671.80710644421.68527733
P112559170.667-0.240138222-2.881658667
P121636496.667-0.393917222-4.727006667
P133100538.667-0.149910222-1.798922667
NC2579499.333-0.236750111-2.841001333
P213109118-0.148480333-1.781764
P222270145.667-0.288309056-3.459708667
P232761933-0.2063445-2.476134

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 21.685 μg/mL
Your negative control PCR result was -2.841 μg/mL

Patient 74963 : The images for this patient had an absence of a reaction from the SYBR green dye, so it was a clear droplet. The final result was -3.136 μg/mL.
Patient 97208 : The images for this patient had an absence of a reaction from the SYBR green dye, so it was a clear droplet. The final result was -2.573 μg/mL.

Patient 74963 : The images are more closely related to the negative control because there was an absence of reaction with the SYBR green dye. The image did not resemble the positive control, the positive control had a green color and the patients was clear. Thus the drop was clear of the target DNA sequence.
Patient 97208 : The images are more closely related to the negative control because there was an absence of reaction with the SYBR green dye. The image did not resemble the positive control, the positive control had a green color and the patients was clear. Thus the drop was clear of the target DNA sequence.

Image J Values for All Samples

Final DNA concentration
in SYBR Green I solution (µg/mL)
AREA
(pixels)
Mean Pixel Value RAWINTDEN
of the drop
RAWINTDEN
of the background
2.550642168.1148513611385684
2.550642166.1938416341409557
2.550642164.3648323742395546
150642114.9385820701360584
150642111.455644030358166
150642117.5985955383370618
0.55064278.4513972933336693
0.55064277.9213946083381415
0.55064286.9874405175349967
0.255064247.1992390227331595
0.255064245.22289040321962
0.255064249.1112487086355290
0.1255064228.4181439120347140
0.1255064230.1821528457423950
0.1255064230.0721522889330093
05064213.81699375335796
05064214.619740329398902
05064214.501734349374002


Fitting a Straight Line

Fluorimetry Calibration with Equation




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