BME103:T930 Group 10 l2

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Revision as of 13:43, 15 November 2012

BME 103 Fall 2012 Home
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Lab Write-Up 1
Lab Write-Up 2
Lab Write-Up 3
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Contents

OUR TEAM

Name: StudentRole(s)
Name: Student
Role(s)
Name: StudentRole(s)
Name: Student
Role(s)
Name: StudentRole(s)
Name: Student
Role(s)
Name: Mikayle Holm Role: Experimental Protocol Planner
Name: Mikayle Holm
Role: Experimental Protocol Planner
Name: StudentRole(s)
Name: Student
Role(s)

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

Materials For PCR Protocol

Supplied in the Kit Volume
PCR Reaction Mix(a.k.a. GoTaq Mix)
Tag DNA Polymerase enough for exp.
MgCl2, dNTP's, Forward and Reverse Primer Ammounts are all suppliedd in kit
DNA Samples 50 μL each
Patients' DNADNA of Rabbit Eyeball
Negative controlPositive Control
SYBR GREEN1' '
Supplied by User Amount
Lab Coat1 or 2
PCR machine made of steel with top that snapsmust clip shut!
Fluorimeter with built in camera and slides with dots farther apart12 new glass slides
micropipettesat least 5 for mistakes
Eppendorf tubesyou will have a whole box, but will need one for each test


PCR Protocol
BE CAREFUL NOT TO CROSS CONTAMINATE!!

1. Label Eppendorf Tubes with 1-17. Each tube should contain 50 microliters of one of the following substances:

Test Tube Contents
1Positive Control
2Negative Control
3Patient 1, Replicate 1
4Patient 1, Replicate 2
5Patient 1, Replicate 3
6Patient 2, Replicate 1
7Patient 2, Replicate 2
8Patient 2, Replicate 3
9Patient 3, Replicate 1
10Patient 3, Replicate 2
11Patient 3, Replicate 3
12Patient 4, Replicate 1
13Patient 4, Replicate 2
14Patient 4, Replicate 3
15Patient 5, Replicate 1
16Patient 5, Replicate 2
17Patient 5, Replicate 3

2. Use properly labeled micropipette to place 17 samples into 17 properly labeled Eppendorf tubes already containing 50 microliters GoTaq mix. To see contents of GoTaq mix, see materials.
3. Place 17 Eppendorf tubes containing DNA and GoTaq Mix in Open PCR Machine. This PCR machine is made of steel in order to decrease safety hazards, and is able to hold more Eppendorf tubes. Therefore, all 17 Eppendorf tubes fit in this PCR. The top of the PCR machine snaps shut and can be clipped down to be less dangerous.
4. To make up for the more Eppendorf tubes in the PCR machine, each cycle is increased by 2 degrees Celsius and more power is needed to increase temperature, making the Thermal Cycler program:
Stage One: 1 cycle, 97 degrees Celsius for 180 seconds
Stage Two: 35 cycles, 97 degrees Celsius for 30 seconds, 59 degrees for 30 seconds, 74 degrees Celsius for 30 seconds
Stage Three: 74 degrees Celsius for 180 seconds
Final Hold: 6 degrees Celsius
5. When PCR is complete, proceed to DNA Measurement Protocol

DNA Measurement Protocol
BE CAREFUL NOT TO CROSS CONTAMINATE!!

1. With permanent marker, clearly number micropipettes. With permanent marker, number Eppendorf tubes at the top. There should be 17 labeled micropipettes and 17 Eppendorf tubes.
2. Transfer each sample from PCR Eppendorf tubes to labeled (1-17) Eppendorf tubes containing 400 microliters of buffer. Get all of the sample into the Eppendorf tubes.
3. Take specially labeled Eppendorf tube (18) with SYBR GREEN I and using specially labeled micropipette (18) place 2 drops on first two centered drops on fluorimeter slide. These dots are further apart on this fluorimeter slide in order to avoid contamination.
4. Take diluted sample from labeled Eppendorf tubes and place 2 drops on top of sample of SYBR GREEN I drop.
5. Turn light on, place beam so that it is passing through the drop.
6. Take picture with built in camera to avoid variability on smart phone camera settings.
7. Use micropipette properly labeled with black strip for waste to discard sample.
8. Repeat 3-7 for all samples (Eppendorf tubes 1-17), the DNA of rabbit eyeball (Eppendorf tube 19), and scintillation vial.
9. Generate pictures in ImageJ to analyze results.

Research and Development

Background on Disease Markers



Primer Design



Illustration


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