Name: Coleen Fox Role: Experimental Protocol Planner
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
After using the Open PCR machine, we noticed a few components of the machine that could be improved. The biggest issue we encountered was that of the lid. When placing the tubes into the machine, it was very difficult to unlatch the lid from the base. Additionally, when screwing the heating plate down onto the tubes, it was difficult to tell how much the plate needed to be screwed down. With these problems in mind, we decided to redesign the lid by replacing two of the panels of the lid with plexi-glass and replace the hinge with magnets. By replacing two of the panels with plexi-glass, the user is able to see the heating plate being screwed down onto the tubes. This ensures that proper placement and rids of possible experimental errors. By replacing the hinge with magnets, we eliminate the difficulty experienced with the hinge. Also, placing magnets at the corners of the lid to hold the lid in place allows for greater visibility when adding the tubes to the tray.
Instructions
Assembly Instructions
1. Same procedure following to lid.
User Instructions
Protocols
MaterialsFor 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' DNA
DNA of Rabbit Eyeball
Negative control
Positive Control
SYBR GREEN1
' '
Supplied by User
Amount
Lab Coat
1 or 2
PCR machine made of steel with top that snaps
must clip shut!
Fluorimeter with built in camera and slides with dots farther apart
12 new glass slides
micropipettes
at least 5 for mistakes
Eppendorf tubes
you 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
1
Positive Control
2
Negative Control
3
Patient 1, Replicate 1
4
Patient 1, Replicate 2
5
Patient 1, Replicate 3
6
Patient 2, Replicate 1
7
Patient 2, Replicate 2
8
Patient 2, Replicate 3
9
Patient 3, Replicate 1
10
Patient 3, Replicate 2
11
Patient 3, Replicate 3
12
Patient 4, Replicate 1
13
Patient 4, Replicate 2
14
Patient 4, Replicate 3
15
Patient 5, Replicate 1
16
Patient 5, Replicate 2
17
Patient 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.
BME 103 Fall 2012
