BME103:T130 Group 7 l2: Difference between revisions
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{| style="wikitable" width="700px" | {| style="wikitable" width="700px" | ||
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| [[Image:EmilyBMElab.jpg|100px|thumb|Name: Emily Thompson<br>Research & Development Scientist]] | | [[Image:EmilyBMElab.jpg|100px|thumb|Name: [http://openwetware.org/wiki/User:Emily_Thompson Emily Thompson]<br>Research & Development Scientist<br>]] | ||
| [[Image: | | [[Image:vivwiki.jpg|100px|thumb|Name: [http://openwetware.org/wiki/User:Vivian_Rose_Benjes Vivian Benjes]<br>Experimental Protocol Planner/ Research & Development<br>]] | ||
| [[Image: | | [[Image:francesfbook.jpg|100px|thumb|Name: Frances Marrett<br>Experimental Protocol Planner/ Editor<br>]] | ||
| [[Image:ChrisBMElab.jpg|100px|thumb|Name: [http://openwetware.org/wiki/User:Christopher_M._Glass Chris Glass]<br>Open PCR Machine Engineer<br>]] | | [[Image:ChrisBMElab.jpg|100px|thumb|Name: [http://openwetware.org/wiki/User:Christopher_M._Glass Chris Glass]<br>Open PCR Machine Engineer/ Experimental Protocol Planner<br>]] | ||
| [[Image:RyanBMElab.jpg|100px|thumb|Name: | | [[Image:RyanBMElab.jpg|100px|thumb|Name: [http://openwetware.org/wiki/User:Ryan_T._Frantz Ryan Frantz]<br>Open PCR Machine Engineer<br>]] | ||
|} | |} | ||
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==Thermal Cycler Engineering== | ==Thermal Cycler Engineering== | ||
Our re-design is based upon the [http://openpcr.org Open PCR] system originally designed by Josh Perfetto and Tito Jankowski.<br> | Our [http://www.youtube.com/watch?v=MJY70uV4qbI&feature=youtu.be re-design] is based upon the [http://openpcr.org Open PCR] system originally designed by Josh Perfetto and Tito Jankowski.<br> | ||
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[[Image:32 wells final.png]] | [[Image:32 wells final.png]] | ||
<br> | <br> | ||
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 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, as well as the size of the lid, so that the samples would be insulated during the PCR reaction. | ||
<br><br><br> | <br><br><br> | ||
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. | 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. | ||
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<br><br> | <br><br> | ||
Longer Power Cord: <br> | Longer Power Cord: <br> | ||
The longer cord will be installed in the same manner that the | The longer cord will be installed in the same manner that the power cord was. | ||
<br><br> | <br><br> | ||
Doubled PCR Blocks:<br> | Doubled PCR Blocks:<br> | ||
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Thermal Cycler for PCR reaction <br> | Thermal Cycler for PCR reaction <br> | ||
Cycling PCR kit (50) <br> | |||
[http://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&vertical=LSE&country=US&lang=en&productID=170-9799EDU BioRad Real Time PCR Applications Guide]<br> | [http://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&vertical=LSE&country=US&lang=en&productID=170-9799EDU BioRad Real Time PCR Applications Guide]<br> | ||
Basic equipment for Fluorimetry <br> | |||
-"Dark Room" box <br> | |||
-Camera stand <br> | |||
-Teflon coated well slides <br> | |||
-Cybergreen solution <br> | |||
-Instructions for use <br> | |||
<br> | <br> | ||
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Access to 120V electrical outlet<br> | Access to 120V electrical outlet<br> | ||
[http://www.sigmaaldrich.com/catalog/product/sigma/z717290?lang=en®ion=US 10 uL pipette]<br> | [http://www.sigmaaldrich.com/catalog/product/sigma/z717290?lang=en®ion=US 10 uL pipette]<br> | ||
[http://www.usascientific.com/80-place-tube-rack-standard-colors.aspx Eppendorf tube block] <br> | [http://www.usascientific.com/80-place-tube-rack-standard-colors.aspx Eppendorf tube block] <br><br> | ||
'''PCR Protocol''' | '''PCR Protocol''' | ||
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'''DNA Measurement Protocol''' | '''DNA Measurement Protocol''' | ||
<br> | |||
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> | |||
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> | |||
- Exposure to highest setting <br> | |||
- ISO to 800+ <br> | |||
- White balance to Automatic <br> | |||
- Saturation to highest setting <br> | |||
- Flash off if necessary <br> | |||
- Contrast to lowest setting <br> | |||
<br><br> | |||
'''Picture Analysis:''' <br> | |||
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:''' <br> | |||
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== | ||
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'''Background on Disease Markers'''<br> | '''Background on Disease Markers'''<br> | ||
Alzheimer's disease, a form of dimentia in which brain function is lost gradually over time, is associated with the SNP [http://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?rs=121918396 rs121918396]. The sequence associated with Alzheimer's is TAG, while a normal sequence is TGG. <br> | Alzheimer's disease, a form of dimentia in which brain function is lost gradually over time, is associated with the SNP [http://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?rs=121918396 rs121918396]. The sequence associated with Alzheimer's is TAG, while a normal sequence is TGG. <br> | ||
'''Primer Design'''<br> | |||
''' | Normal bp sequence:<br> | ||
GGCCCAGGCC'''TAG'''GGCGAGCGGCT<br> | |||
< | bp sequence with Alzheimer's associated SNP: <br> | ||
GGCCCAGGCC'''TGG'''GGCGAGCGGCT <br> | |||
To detect the SNP associated with Alzheimer's, two primers are necessary: one forward primer and one reverse primer. The forward primer matches the top strand sequence GCCCAGGCCTAGGGCGAGCGGC. The primer binds to the bottom strand because the bottom strand has corresponding base pairs. The reverse primer located about 200bp away from the forward primer matches the sequence on the bottom strand, AACTCTTACACTGCATACAT. This primer binds to the top strand and prepares for DNA Polymerase to build a new bottom strand. The two primers are useful in the second step of PCR, when the temperature drops to 57 degrees Celsius and primers bind around the target sequence. The primer design allows the target sequence to be replicated and multiplied millions of times through PCR. If only the forward primer was being used, the DNA sequence would be replicated, but not amplified. <br> | |||
'''Illustration''' | '''Illustration''' | ||
[[Image:EmilyPCRpic.jpg|700px]]<br> | |||
Adapted from: http://users.ugent.be/~avierstr/principles/pcr.html <br><br><br> | |||
<!--- Include an illustration that shows how your system's primers allow specific amplification of the disease-related SNP ---> | <!--- Include an illustration that shows how your system's primers allow specific amplification of the disease-related SNP ---> | ||
Latest revision as of 12:20, 5 December 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 | |||||
OUR TEAMLAB 2 WRITE-UPThermal Cycler EngineeringOur re-design is based upon the Open PCR system originally designed by Josh Perfetto and Tito Jankowski.
ProtocolsMaterials
Thermal Cycler for PCR reaction Supplied by User Sterile gloves PCR Protocol Polymerase Chain Reaction
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 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 DevelopmentBackground on Disease Markers
Primer Design Normal bp sequence: bp sequence with Alzheimer's associated SNP:
|