BME103:T130 Group 3 l2

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(Protocols)
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==Protocols==
==Protocols==
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<!--- Design a new protocol based on your group's new PCR design. Make a step-by-step list of how someone should use your method
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<font size=3><b>Polymerase Chain Reaction</b></font><br><br>
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Things to consider:
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PCR is a method of amplifying a sample of DNA. PCR alters the temperature and allows the DNA to separate, bind to primers, and catalyze. This results in the amount of DNA doubling after each cycle. <br>
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How should the PCR machine be set up? Does it need to be plugged in?
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<b>Procedure:</b><br>
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How many samples will fit into a single 2-hour run?
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1.) The DNA samples were heated to ninety-five degrees Celsius (95°C) for three (3) minutes to unzip the two single strands. <br>
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How many replicates should be created per patient?
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2.) They were then cooled to fifty-seven degrees Celsius (57°C) and the primers were attached to their matching sequences. <br>
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What should the final volume of the reaction be?
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3.) They were then heated back to seventy-two degrees Celsius (72°C) and polymerase extended the DNA strands by attaching the correct free nucleotides in order on the single strands. <br><br>
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Will signal reading be integrated into the PCR machine or remain separate?
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If it is separate, you will need to include instructions on how to use the fluorimeter.
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How should the user calculate the about of signal amplified?
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etc.
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--->
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'''Materials'''
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<b>GoTaq Mix Components For 100μl reaction volume:</b><br>
 +
2X GoTaq Colorless Master Mix<br>
 +
10 μM upstream primer<br>
 +
10 μM downstream primer<Br>
 +
DNA template<br>
 +
Nuclease-Free Water to<Br><br>
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<!--- Place your two tables "Supplied in the kit" and "Supplied by User" here --->
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{| {{table}}
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| align="center" style="background:#f0f0f0;"|'''Reagent'''
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| align="center" style="background:#f0f0f0;"|'''Volume'''
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|-
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| Template DNA (20 ng)||0.1μL
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|-
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| 10μM forward primer||0.5μL
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|-
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| 10μM reverse primer||0.5μL
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|-
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| GoTaq master mix||25.0μL
 +
|-
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| dH2O||23.9μL
 +
|-
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| Total Volume||50.0μL
 +
|}
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'''PCR Protocol'''
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Patient 1<br>
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ID 30269<br>
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Male, 55 years old
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Patient 2<br>
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ID 22057<br>
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Female, 55 years old<br>
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[[Image:Flourimeter Group 3.jpg|600x300px]]<br><br>
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<font size=3>'''Fluorimeter Setup'''</font><br><br>
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1.) The lid was first taken off the the box and one of its sides was unbuttoned in order to create a flap.<br>
 +
2.) The box was the flipped upside down in order to create a dark environment for the camera.<br>
 +
3.) A hydrophobic slide was then inserted into the flourimeter.<br>
 +
4.) Finally, the camera phone was placed in the stand.
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'''DNA Measurement Protocol'''
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<font size=3>'''Fluorimeter Measurements'''</font><br><br>
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<b>Fluorimeter Assembly Procedure</b><br>
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1.) Label transfer pipettes and tubes<br>
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2.) Transfer each sample separately into tube containing 400μl of buffer<Br>
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3.) Take the specifically labeled tube containing SYBR GREEN 1 and place 2 drops on the first 2 centered drops <br>
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4.) Place 2 drops of diluted sample on top of the SYBR GREEN 1 drop<br>
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5.) Align light through drop<br>
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6.) Take pictures using light box<br>
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7.) Repeat for each sample.<br>
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8.) Run water as BLANK using same procedure<br>
 +
<b>ImageJ Instructions </b><br>
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1.) Open ImageJ<br>
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2.) Click <b>ANALYZE</b> tool bar and select <b>SET MEASUREMENTS</b><br>
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3.) Select <b>AREA, MEAN GREY VALUE,</b> and<b> INTEGRATED DENSITY</b><br>
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4.) Upload image to ImageJ<br>
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5.) Select <b>IMAGE</b> then <b>COLOR</b> and then <b> SPLIT CHANNELS</b><br>
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6.) Only use green channel<br>
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7.) Use <b>OVAL</b> tool and select the entire drop of liquid<br>
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8.) Go to <b> ANALYZE</b> and then <b>MEASURE</b><br>
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9.) Drag circle to the background of the image <br>
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10.) Record results <br>
 +
11.) Repeat if necessary
 +
<br><br>
==Research and Development==
==Research and Development==

Revision as of 16:57, 15 November 2012

BME 103 Fall 2012 Home
People
Lab Write-Up 1
Lab Write-Up 2
Lab Write-Up 3
Course Logistics For Instructors
Photos
Wiki Editing Help
Image:BME494_Asu_logo.png

Contents

OUR TEAM

Name: Serena KaplanResearch and Development
Name: Serena Kaplan
Research and Development
Name: Gabe McInnisOpen PCR Machine Engineer
Name: Gabe McInnis
Open PCR Machine Engineer
Name: Blake EichlerExperimental Protocol Planner
Name: Blake Eichler
Experimental Protocol Planner
Name: Sierra MorrisExperimental Protocol Planner
Name: Sierra Morris
Experimental Protocol Planner
Name: Zazu MoloiOpen PCR Machine Engineer
Name: Zazu Moloi
Open PCR Machine Engineer
Name: Katelin VaughnResearch and Development
Name: Katelin Vaughn
Research and Development

LAB 2 WRITE-UP

Thermal Cycler Engineering

Our re-design is based upon the Open PCR system originally designed by Josh Perfetto and Tito Jankowski.


System Design


Key Features


Instructions





Protocols

Polymerase Chain Reaction

PCR is a method of amplifying a sample of DNA. PCR alters the temperature and allows the DNA to separate, bind to primers, and catalyze. This results in the amount of DNA doubling after each cycle.
Procedure:
1.) The DNA samples were heated to ninety-five degrees Celsius (95°C) for three (3) minutes to unzip the two single strands.
2.) They were then cooled to fifty-seven degrees Celsius (57°C) and the primers were attached to their matching sequences.
3.) They were then heated back to seventy-two degrees Celsius (72°C) and polymerase extended the DNA strands by attaching the correct free nucleotides in order on the single strands.

GoTaq Mix Components For 100μl reaction volume:
2X GoTaq Colorless Master Mix
10 μM upstream primer
10 μM downstream primer
DNA template
Nuclease-Free Water to

Reagent Volume
Template DNA (20 ng)0.1μL
10μM forward primer0.5μL
10μM reverse primer0.5μL
GoTaq master mix25.0μL
dH2O23.9μL
Total Volume50.0μL


Patient 1
ID 30269
Male, 55 years old

Patient 2
ID 22057
Female, 55 years old



Fluorimeter Setup

1.) The lid was first taken off the the box and one of its sides was unbuttoned in order to create a flap.
2.) The box was the flipped upside down in order to create a dark environment for the camera.
3.) A hydrophobic slide was then inserted into the flourimeter.
4.) Finally, the camera phone was placed in the stand.

Fluorimeter Measurements

Fluorimeter Assembly Procedure
1.) Label transfer pipettes and tubes
2.) Transfer each sample separately into tube containing 400μl of buffer
3.) Take the specifically labeled tube containing SYBR GREEN 1 and place 2 drops on the first 2 centered drops
4.) Place 2 drops of diluted sample on top of the SYBR GREEN 1 drop
5.) Align light through drop
6.) Take pictures using light box
7.) Repeat for each sample.
8.) Run water as BLANK using same procedure


ImageJ Instructions
1.) Open ImageJ
2.) Click ANALYZE tool bar and select SET MEASUREMENTS
3.) Select AREA, MEAN GREY VALUE, and INTEGRATED DENSITY
4.) Upload image to ImageJ
5.) Select IMAGE then COLOR and then SPLIT CHANNELS
6.) Only use green channel
7.) Use OVAL tool and select the entire drop of liquid
8.) Go to ANALYZE and then MEASURE
9.) Drag circle to the background of the image
10.) Record results
11.) Repeat if necessary

Research and Development

Background on Disease Markers



Primer Design



Illustration


Personal tools