SYBR Green Dye
SYBR Green Dye is essentially a dye that turns DNA green which helps in detecting DNA is a DNA fluorimeter.
Single-Drop Fluorimeter
The Single-Drop Fluorimeter is a small plastic box with a apparatus located on the top where the multi-welled slide fits. It has a switch on the right hand side for the LED light to be turned on or off. The slide of the fluorometer is positioned in such a way that the blue LED shines through a drop placed over two middle wells. The slide has holes along it so that a sample can be placed in them.
How the Fluorescence Technique Works
The Fluorimeter has a slide with small holes where drops of sample DNA were placed. The drops remain in their round shape due to a Teflon layer on the slide. The Teflon gives the slide texture and allows the sample drop to retain the "drop" shape. The reason this is important is because the light from the fluorimeter can go pierce through the drop in order to increase the intensity of the DNA glow. This also allows for the SYBR Green to migrate to the surface, which makes the glow seen on the top of the drop.This all makes it easier to view and analyze whether the sample has a green glow, and whether or not there was DNA present in the sample.
Procedure
Smart Phone Camera Settings
Type of Smartphone: iPhone 5s
Flash: N/A
ISO setting: N/A
White Balance: N/A
Exposure:N/A
Saturation: N/A
Contrast: N/A
Calibration
Place the smart phone on the cradle at a right angle from the slide. Adjust the height of the fluorimeter using the plastic trays so that the camera takes a picture of the sample drop sideways.
Adjust the distance between the smartphone on its cradle and the first two rows of the slide so that it is as close as you can get without having a blurry image. Record the distance between the smart phone cradle and drop using the ruler provided.
Distance between the smart phone cradle and drop = 8 cm.
Solutions Used for Calibration
Initial Concentration of 2x Calf Thymus DNA solution (µg /mL)
INTDEN
.125
345337
.5
509104
2.5
1121876
Placing Samples onto the Fluorimeter
Step 1: Set your micropipette to 80 microliters and place a 80 microliter drop of SYBR GREEN I in the middle of the first two rows of the slide. Add 80 microliters of one of the 5 µg /mL Calf Thymus DNA solution. This is now a "sample".
Step 2: Align the drop by moving the slide so that the blue LED light is focused by the drop to the middle of the black fiber optic fitting.
Step 3: Use the timer on your camera to take a picture after covering the fluorimeter and camera with the light box. Take 1 image of the drop.
Step 4: Remove the box and be sure not to move your smartphone. If you want to adjust for any movement, use the “distance between the smart phone camera and drop” that you recorded.
Step 5: Use the pipette to remove the 160 µL drop from the surface and move the slide to the next position. Take at least one measurement of a “sample” of the same concentration of DNA in one of the slide positions, remove the drop, move the slide to the next position, and then use the next concentration standard.
Step 6: Repeat steps 3 – 8 for just two more of the Calf Thymus DNA solutions.
Step 7: ImageJ: For each image, draw a circle around the drop and measure it the same way as last week’s lab, but do not measure the background noise.
Step 8: Data processing: Use just the RAWINTDEN OF THE DROP to plot a new calibration curve. Add a line of best fit. You will need this line and its equation later.
Data Analysis
Representative Images of Samples
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.]
Initial Concentration of 2X Calf Thymus DNA solution (µg /mL)
Volume of the 2X DNA solution (µL)
Volume of the SYBR GREEN I
Dye solution (µL)
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
Fitting a Straight Line
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: 12.11 μg/mL
Your negative control PCR result was: 0.75 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 51225 : We pbserved a green color for the patient. Indicating the samples were positive.
Patient 60779 : We observed a blue color for the patient. Indicating the samples were negative.
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 51225 : The patient's sample turned out to be positive because of the green color given off. Indicating that the patient does not have the disease.
Patient 60779 : The patient's sample turned out to be negative because of the blue color given off. Indicating that the patient has the disease.
BME 100 Spring 2014
