BME103:T130 Group 7 l2

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'''DNA Measurement Protocol'''
'''DNA Measurement Protocol'''
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To measure the DNA production during the PCR a fluorimeter will be employed as before. Cybergreen will be added to the DNA sample from the PCR machine and this will cause a faint fluorescence in the sample. While this will not be detectable to the human eye, a camera of moderate quality, such as one in a smart phone will be capable of capturing the faint glow. The digital image from the camera will then need to be analyzed with the open source program Imagej. This software is free to download and easy enough for students to use.
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To analyze the DNA samples after they've been amplified, a fluorimeter can be used. The Fluorimeter aparatus consists of a hydrophobic Teflon surface on a glass slide. There is an array of 3x10 glass wells on the surface to anchor drops in position for photographing. First, two drops of the cybergreen solution should be placed with a sterile pipet. Then two drops of amplified sample DNA should be added to the cybergreen solution with another sterile pipet. It is important to place the DNA on top of the cybergreen in order to achieve the best results and to avoid contamination of the DNA samples. The drops should then be photographed in the provided dark box with the door shut to eliminate excess background light. The slide was then moved back by two columns for the next sample. This procedure can be replicated for each of the samples. <br> <br>
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A smartphone with adjustable settings can be used to take a photo of the samples. To achieve the best results the following settings should be used.<br>
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- Exposure to highest setting <br>
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- ISO to 800+ <br>
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- White balance to Automatic <br>
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- Saturation to highest setting <br>
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- Flash off if necessary <br>
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- Contrast to lowest setting <br>
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<br><br>
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'''Picture Analysis:''' <br><br>
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The pictures of each sample should be analyzed using the "image j" software. This software is open source and can be obtained free of charge. The pictures, taken with the settings listed above, can be uploaded to the "image j" software then the images can be separated into their composite colors, red, blue, and green. The green portion of the images can then analyzed using the analyze option in the software and its "INTDEN" can be recorded.
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'''Interpreting results:''' <br><br>
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The cybergreen solution allows the amount of DNA in a sample to be determined by its "INTDEN". The cybergreen glowed green in the presence of DNA and while this glow is not visable to the naked eye, the smart phone in conjunction with "image j" is able to quantify the luminosity in terms of the "INTDEN". The brighter samples contain more DNA and have higher "INTDEN" value. This indicates that the sample reacted with the primers better. By providing positive and negative controls in the experiment the various samples can be compared and their reaction to the PCR primer can be determined.
==Research and Development==
==Research and Development==

Revision as of 17:41, 28 November 2012

BME 103 Fall 2012 Home
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Image:BME494_Asu_logo.png

Contents

OUR TEAM

Name: Emily ThompsonResearch & Development Scientist
Name: Emily Thompson
Research & Development Scientist
Name: Vivian BenjesExperimental Protocol Planner/ Data Analyst
Name: Vivian Benjes
Experimental Protocol Planner/ Data Analyst
Name: Frances MarrettExperimental Protocol Planner
Name: Frances Marrett
Experimental Protocol Planner
Name: Chris GlassOpen PCR Machine Engineer
Name: Chris Glass
Open PCR Machine Engineer
Name: Ryan FrantzOpen PCR Machine Engineer[1]
Name: Ryan Frantz
Open PCR Machine Engineer
[1]
Name: Cenric NigburGhost/ TBD
Name: Cenric Nigbur
Ghost/ TBD

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

Correctly positioning the heating plate on the plastic sample containers proved to be difficult. To make this product more user friendly a button would be added to the heating plate. When in contact with the samples the button would signal the user via the LED display that the heating lid was correctly positioned.
Image:Roof Assembly small.png
This picture of the roof assembly shows where the button will be added. The blue circle would contain the button that would, when in contact with the plastic containers below, signal to the user that the heating plate was correctly positioned.
Image:Plate with Button.png
This is another image of where the button would be positioned. The relative size is also accurate as it would need to be able to detect a sample anywhere in the heating block.
Image:Plastic Body Final.png
The original PCR machine was make of lightweight balsa wood. While this was inexpensive it was prone to splitting along the grain and staining. The new PCR machine would be made with a stronger plastic body that would better resist wear and tear and resist becoming stained with liquids commonly seen in a lab.
Image:Power Cord Final.png
The original PCR machine had a cord that was slightly short and required an extension cord in some cases. The new PCR machine would have a longer cord so as to avoid this problem.
Image:32 wells final.png
The original PCR machine had a limited storage capacity that proved to be a problem in our large class. To make the product more attractive to educators the new machine would have double the capacity. To help minimize the cost of this addition the new product would use the same PCR blocks as before, merely double the number. To accommodate the extra load the heater and cooler would likewise need to be doubled.


The original PCR machine was slightly difficult to understand, again to make the product more attractive to educators the new product would have key parts engraved with their names. This would allow a student to quickly identify pieces and deduce their function. No picture is shown for this proposed addition.


Key Features
1.Button to display when the heating lid is correctly positioned
2.New plastic housing to replace wood
3.Longer Power Cord
4.Doubled storage capacity
5.Key parts engraved with names (for educational purposes)


Instructions
Heating Plate Button:
This device will need to be attached to the heating plate with provided metal hardware to resist the heat generated. The button will have leads that connect to the main CPU and allow the button to display when the heating lid is in place

Plastic Housing:
The new housing will be installed in the same manner that the old housing was.

Longer Power Cord:
The longer cord will be installed in the same manner that the old housing was.

Doubled PCR Blocks:
This addition will require two PCR blocks to be installed instead of only one. Both will connect to the same CPU and carry out the same function at the same time however. The new PCR block will require the addition of a new heater and cooling unit that will be installed in the same manner that the old units were installed.

Engraved Parts:
These parts will be installed in the same manner that the old parts were.






Protocols

Materials


Supplied in the Kit

Thermal Cycler for PCR reaction
Qiagen Fast Cycling PCR kit (50)
BioRad Real Time PCR Applications Guide


Supplied by User

Sterile gloves
Access to 120V electrical outlet
10 uL pipette
Eppendorf tube block

PCR Protocol

Polymerase Chain Reaction
To amplify samples of DNA, the OpenPCR machine was used to perform a Polymerase Chain Reaction (PCR). This technique worked by cycling a mixture of DNA Template, Primers, Taq Polymerase, Magnesium Chloride, and dNTP's through three specific temperatures to create more copies of the desired sequence. After assembling the PCR mixture, the PCR machine was programmed to perform three stages. In the first stage, the samples went through one cycle at 95⁰C for 3 minutes. The purpose of this stage was to initially denature the DNA and allow the primers to act on the DNA. The second stage put the samples through 35 cycles of 95⁰C, 57⁰C, and 72⁰C each for 30 seconds. The purpose of the first part of the second stage is to break apart the hydrogen bonds between the base pairs, denaturing the DNA sequence into two separate strands. The purpose of the low temperature is to allow primers to bind. The purpose of the middle temperature is to create an environment for Taq Polymerase to assemble a new strand that is the desired product of the entire polymerase chain reaction. The last stage, stage three, puts the samples through one cycle of 72⁰C for 3 minutes. There is a final hold of 4⁰C that preserves the DNA. The samples were then taken out of the PCR machine. The target sequence had been amplified a million times and could now be analyzed with less sensitive equipment! To analyze the sequence, see the Fluorimeter section.


DNA Measurement Protocol

To analyze the DNA samples after they've been amplified, a fluorimeter can be used. The Fluorimeter aparatus consists of a hydrophobic Teflon surface on a glass slide. There is an array of 3x10 glass wells on the surface to anchor drops in position for photographing. First, two drops of the cybergreen solution should be placed with a sterile pipet. Then two drops of amplified sample DNA should be added to the cybergreen solution with another sterile pipet. It is important to place the DNA on top of the cybergreen in order to achieve the best results and to avoid contamination of the DNA samples. The drops should then be photographed in the provided dark box with the door shut to eliminate excess background light. The slide was then moved back by two columns for the next sample. This procedure can be replicated for each of the samples.

A smartphone with adjustable settings can be used to take a photo of the samples. To achieve the best results the following settings should be used.

- Exposure to highest setting
- ISO to 800+
- White balance to Automatic
- Saturation to highest setting
- Flash off if necessary
- Contrast to lowest setting


Picture Analysis:

The pictures of each sample should be analyzed using the "image j" software. This software is open source and can be obtained free of charge. The pictures, taken with the settings listed above, can be uploaded to the "image j" software then the images can be separated into their composite colors, red, blue, and green. The green portion of the images can then analyzed using the analyze option in the software and its "INTDEN" can be recorded.

Interpreting results:

The cybergreen solution allows the amount of DNA in a sample to be determined by its "INTDEN". The cybergreen glowed green in the presence of DNA and while this glow is not visable to the naked eye, the smart phone in conjunction with "image j" is able to quantify the luminosity in terms of the "INTDEN". The brighter samples contain more DNA and have higher "INTDEN" value. This indicates that the sample reacted with the primers better. By providing positive and negative controls in the experiment the various samples can be compared and their reaction to the PCR primer can be determined.

Research and Development

Background on Disease Markers
Alzheimer's disease, a form of dimentia in which brain function is lost gradually over time, is associated with the SNP rs121918396. The sequence associated with Alzheimer's is TAG, while a normal sequence is TGG.



Primer Design



Illustration


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