BME103:T130 Group 10 l2

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(Thermal Cycler Engineering)
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| [[Image:BME103_Group10_FrancesL.jpg|100px|thumb|Name:<br> '''Frances Lakers'''<br>Role:<br>'''Protocol Planner''']]
| [[Image:BME103_Group10_FrancesL.jpg|100px|thumb|Name:<br> '''Frances Lakers'''<br>Role:<br>'''Protocol Planner''']]
| [[Image:BME103_Group10_tylert.jpg|100px|thumb|Name:<br> '''Tyler Tamasauckas'''<br>Role:<br>'''Protocol Planner''']]
| [[Image:BME103_Group10_tylert.jpg|100px|thumb|Name:<br> '''Tyler Tamasauckas'''<br>Role:<br>'''Protocol Planner''']]
-
| [[Image:BME103_Group10_JefferyWhyYouNoUploadProfilePic.jpg|100px|thumb|Name:<br> '''Jeffery Ramirez'''<br>Role:<br>'''R&D Specialist''']]
+
| [[Image:Jeffrey.jpeg|100px|thumb|Name:<br> '''Jeffery Ramirez'''<br>Role:<br>'''R&D Specialist''']]
-
| [[Image:BME103_Group10_AlexWhyYouNoUploadProfilePic.jpg|100px|thumb|Name:<br> '''Alexander Baldwin '''<br>Role:<br>'''Machine Design''']]
+
| [[Image:Monster.jpg|100px|thumb|Name:<br> '''Alexander Baldwin '''<br>Role:<br>'''Machine Design''']]
|}
|}
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'''System Design'''<br>
'''System Design'''<br>
-
 
+
[[Image:BME 103 Group 10 Body Side.png|thumb|center|296px|The casing for the device is a durable plastic, instead of the original's plywood.  This would be instituted across the entire machine.]]   
'''Key Features'''<br>
'''Key Features'''<br>
-
There are two specific features that will be upgraded in the OpenPCR machine - the case and the power source.  The case will be changed from a thin wood to a more durable plastic, so that the device can be taken out into the field.  The device will also have solar panels, which will provide for the power.  This shift allows the device to be used where electricity is not always available, such as rural and undeveloped regions.  When combined, these two features open the device up to a larger market.
+
There are two specific features that will be upgraded in the OpenPCR machine - the case and the power source.  The case will be changed from a thin wood to a more durable plastic, so that the device can be taken out into the field.  The device will also have solar panels, which will provide for the power.  This shift allows the device to be used where electricity is not always available, such as rural and undeveloped regions.  When combined, these two features open the device up to a larger market. <br>
 +
<br>
 +
In addition to the Thermal Cycler's modifications, there have been improvements on the process of fluorimeter image collection and measurement. Instead on manually adjusting phone settings and using a computer with ImageJ to measure colors, an app has been implemented that automatically adjusts the cameras settings to the correct exposure, as well as having the capability to measure the colors right from the smart phone. <br>
'''Instructions'''<br>
'''Instructions'''<br>
-
The instructions would remain the same, as the solar panel array would come attached to the plastic on the device's top side.
+
The instructions would remain mostly the same, as the solar panel array would come attached to the plastic on the device's top side. The only changes in procedure would be in the use of an all in one phone application for the fluorimeter measurements as well as the thermal cycling set-up.
<!--- From Week 4 exercise --->
<!--- From Week 4 exercise --->
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'''Materials'''
'''Materials'''
-
 
+
<br><br>
-
<!--- Place your two tables "Supplied in the kit" and "Supplied by User" here --->
+
-
 
+
-
 
+
-
'''PCR Protocol''' <br>
+
Supplied in Kit
Supplied in Kit
<table border="1" bordercolor="#000000" style="background-color:#FFFFFF" width="50%" cellpadding="3" cellspacing="3">
<table border="1" bordercolor="#000000" style="background-color:#FFFFFF" width="50%" cellpadding="3" cellspacing="3">
Line 68: Line 66:
<tr>
<tr>
-
<td>Item</td>
+
<td>''Item''</td>
-
<td>Quantity</td>
+
<td>''Quantity''</td>
</tr>
</tr>
-
</table><br>
+
<tr>
 +
<td>PCR Machine</td>
 +
<td>1 unit</td>
 +
</tr>
 +
<tr>
 +
<td>Camera Phone Stand</td>
 +
<td>1 unit</td>
 +
</tr>
 +
<tr>
 +
<td>Fluorimeter Stand (LED Light)</td>
 +
<td>1 unit</td>
 +
</tr>
 +
<tr>
 +
<td>BlackOut Box</td>
 +
<td>1 unit</td>
 +
</tr>
 +
<tr>
 +
<td>Operator's Manual</td>
 +
<td>1 unit</td>
 +
</tr>
 +
</table>
 +
Also included: Access code for smart phone app download. <br><br>
Supplied by User
Supplied by User
<table border="1" bordercolor="#000000" style="background-color:#FFFFFF" width="50%" cellpadding="3" cellspacing="3">
<table border="1" bordercolor="#000000" style="background-color:#FFFFFF" width="50%" cellpadding="3" cellspacing="3">
Line 77: Line 96:
<tr>
<tr>
-
<td>Item</td>
+
<td>''Item''</td>
-
<td>Quantity</td>
+
<td>''Quantity''</td>
</tr>
</tr>
 +
<tr>
 +
<td>Smart Phone (w/ Camera)</td>
 +
<td>1 unit</td>
 +
</tr>
 +
<tr>
 +
<td>USB/Phone Cord (if desired)</td>
 +
<td>1 unit</td>
 +
</tr>
 +
<tr>
 +
<td>Micropipettes</td>
 +
<td>(as needed)</td>
 +
</tr>
 +
<tr>
 +
<td>Hydrophobic Glass Slides</td>
 +
<td>(as needed)</td>
 +
</tr>
 +
<tr>
 +
<td>Eppendorf Tubes</td>
 +
<td>Max 16 tubes/trial</td>
 +
</tr>
 +
<tr>
 +
<td>Sample DNA</td>
 +
<td>0.2μL/tube; 16 tubes/trial</td>
 +
</tr>
 +
<tr>
 +
<td>GoTAQ Master Mix</td>
 +
<td>50μL/tube; 16 tubes/trial</td>
 +
</tr>
 +
<tr>
 +
<td>10 μM Forward Primer</td>
 +
<td>1μL/tube; 16 tubes/trial</td>
 +
</tr>
 +
<tr>
 +
<td>10 μM Reverse Primer</td>
 +
<td>1μL/tube; 16 tubes/trial</td>
 +
</tr>
 +
<tr>
 +
<td>Distilled H<sub>2</sub>O</td>
 +
<td>47.8μL/tube; 16 tubes/trial</td>
 +
</tr>
 +
<tr>
 +
<td>SYBR Green</td>
 +
<td>0.1mL/test sample</td>
 +
</tr>
</table><br>
</table><br>
-
'''DNA Measurement Protocol'''
+
 
 +
'''PCR Protocol'''
 +
<br>
 +
1.) Be sure to be in an area with natural lighting for use of the solar battery, or locate an area with a standard power source availible <br>
 +
2.) Connect the PCR machine via USB or Bluetooth to the phone <br>
 +
4.) Label tubes that will contain the subject DNA. Also be sure to include control tubes in this labeling. <br>
 +
5.) After filling the tubes with sample DNA, primers and other reactants. <br>
 +
6.) Load the tubes into the Thermal cycler's tray. <br>
 +
7.) Close and properly tighten the lid. <br>
 +
8.) Adjust the settings for the thermal cycler. <br>
 +
9.) Begin the program. <br>
 +
 
 +
'''DNA Measurement Protocol''' <br>
 +
1.) Assemble the fluorimeter and place the glass slide in the tray. <br>
 +
2.) Use the pipette to add to drops of water to the slide. These will serve as controls for the measurements. <br>
 +
3.) Adjust the slide to be aligned with the blue LED. The LED should be shining directly on the test drops. <br>
 +
4.) Open the newly introduced all in one fluorimeter application on the smartphone. The application will automatically set the phones camera to the correct settings. <br>
 +
5.) Place the smartphone in the holder and use the in app photo timer to take the picture while the black out box is closed. <br>
 +
6.) Analyze the images color measurements using the app.
 +
7.) Save the image properties as the control image for future measurements. <br>
 +
8.) Repeat steps 2 through 6 for each DNA sample. <br>
 +
9.) The app will automatically compare the control images to the sample images and will list the results. <br>
==Research and Development==
==Research and Development==
Line 91: Line 175:
<!--- A description of the diseases and their associated SNP's (include the database reference number and web link) --->
<!--- A description of the diseases and their associated SNP's (include the database reference number and web link) --->
 +
The disease that we chose was Sickle Cell Anemia. Sickle Cell is a inherited disease that comes from both parents. If only one parent has the Sickle Cell trait, then the child will be a carrier. If both parents have the trait, then the child will have Sickle Cell. This disease causes the red blood cells to change into a crescent shape instead of being disk shape resulting in the loss of efficient oxygen delivery. It can also result in clotting because the blood cells can not make it through small blood vessels very well. The SNP for this disease is 78478128 and the reference SNP is rs78478128.
 +
The link to the page with more info on Sickle cell is  http://www.ncbi.nlm.nih.gov/pubmedhealth/PMH0001554/
 +
 +
The link to the page with the genetic information and SNP is  http://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?rs=78478128#locus
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<!--- Include the sequences of your forward and reverse primers. Explain why a disease allele will give a PCR product and the non-disease allele will not. --->
<!--- Include the sequences of your forward and reverse primers. Explain why a disease allele will give a PCR product and the non-disease allele will not. --->
 +
The forward primer for Sickle Cell is 5'-tcctaagccagtgccagaag-3'
 +
 +
The reverse primer for Sickle Cell is 5'-gaattcgtctgtttcccattctaaac-3'
 +
 +
The mutation looks like this: TCCTG'''A'''GGAG with the bold a being the mutation.
 +
 +
A disease allele will give a PCR result and a non-disease allele will not because PCR is designed to amplify the area that corresponds to the mutation on the DNA. If it did not amplify only the target region, there would be too much information and a result would not be possible.
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<!--- 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 --->
 +
 +
[[Image:PCR_process.gif]]
<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->
<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->
|}
|}

Current revision

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: Frances LakersRole:Protocol Planner
Name:
Frances Lakers
Role:
Protocol Planner
Name: Tyler TamasauckasRole:Protocol Planner
Name:
Tyler Tamasauckas
Role:
Protocol Planner
Name: Jeffery RamirezRole:R&D Specialist
Name:
Jeffery Ramirez
Role:
R&D Specialist
Name: Alexander Baldwin Role:Machine Design
Name:
Alexander Baldwin
Role:
Machine Design

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

The casing for the device is a durable plastic, instead of the original's plywood.  This would be instituted across the entire machine.
The casing for the device is a durable plastic, instead of the original's plywood. This would be instituted across the entire machine.


Key Features

There are two specific features that will be upgraded in the OpenPCR machine - the case and the power source. The case will be changed from a thin wood to a more durable plastic, so that the device can be taken out into the field. The device will also have solar panels, which will provide for the power. This shift allows the device to be used where electricity is not always available, such as rural and undeveloped regions. When combined, these two features open the device up to a larger market.

In addition to the Thermal Cycler's modifications, there have been improvements on the process of fluorimeter image collection and measurement. Instead on manually adjusting phone settings and using a computer with ImageJ to measure colors, an app has been implemented that automatically adjusts the cameras settings to the correct exposure, as well as having the capability to measure the colors right from the smart phone.

Instructions

The instructions would remain mostly the same, as the solar panel array would come attached to the plastic on the device's top side. The only changes in procedure would be in the use of an all in one phone application for the fluorimeter measurements as well as the thermal cycling set-up.




Protocols

Materials

Supplied in Kit

Item Quantity
PCR Machine 1 unit
Camera Phone Stand 1 unit
Fluorimeter Stand (LED Light) 1 unit
BlackOut Box 1 unit
Operator's Manual 1 unit

Also included: Access code for smart phone app download.

Supplied by User

Item Quantity
Smart Phone (w/ Camera) 1 unit
USB/Phone Cord (if desired) 1 unit
Micropipettes (as needed)
Hydrophobic Glass Slides (as needed)
Eppendorf Tubes Max 16 tubes/trial
Sample DNA 0.2μL/tube; 16 tubes/trial
GoTAQ Master Mix 50μL/tube; 16 tubes/trial
10 μM Forward Primer 1μL/tube; 16 tubes/trial
10 μM Reverse Primer 1μL/tube; 16 tubes/trial
Distilled H2O 47.8μL/tube; 16 tubes/trial
SYBR Green 0.1mL/test sample

PCR Protocol
1.) Be sure to be in an area with natural lighting for use of the solar battery, or locate an area with a standard power source availible
2.) Connect the PCR machine via USB or Bluetooth to the phone
4.) Label tubes that will contain the subject DNA. Also be sure to include control tubes in this labeling.
5.) After filling the tubes with sample DNA, primers and other reactants.
6.) Load the tubes into the Thermal cycler's tray.
7.) Close and properly tighten the lid.
8.) Adjust the settings for the thermal cycler.
9.) Begin the program.

DNA Measurement Protocol
1.) Assemble the fluorimeter and place the glass slide in the tray.
2.) Use the pipette to add to drops of water to the slide. These will serve as controls for the measurements.
3.) Adjust the slide to be aligned with the blue LED. The LED should be shining directly on the test drops.
4.) Open the newly introduced all in one fluorimeter application on the smartphone. The application will automatically set the phones camera to the correct settings.
5.) Place the smartphone in the holder and use the in app photo timer to take the picture while the black out box is closed.
6.) Analyze the images color measurements using the app. 7.) Save the image properties as the control image for future measurements.
8.) Repeat steps 2 through 6 for each DNA sample.
9.) The app will automatically compare the control images to the sample images and will list the results.

Research and Development

Background on Disease Markers

The disease that we chose was Sickle Cell Anemia. Sickle Cell is a inherited disease that comes from both parents. If only one parent has the Sickle Cell trait, then the child will be a carrier. If both parents have the trait, then the child will have Sickle Cell. This disease causes the red blood cells to change into a crescent shape instead of being disk shape resulting in the loss of efficient oxygen delivery. It can also result in clotting because the blood cells can not make it through small blood vessels very well. The SNP for this disease is 78478128 and the reference SNP is rs78478128.

The link to the page with more info on Sickle cell is http://www.ncbi.nlm.nih.gov/pubmedhealth/PMH0001554/

The link to the page with the genetic information and SNP is http://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?rs=78478128#locus


Primer Design

The forward primer for Sickle Cell is 5'-tcctaagccagtgccagaag-3'

The reverse primer for Sickle Cell is 5'-gaattcgtctgtttcccattctaaac-3'

The mutation looks like this: TCCTGAGGAG with the bold a being the mutation.

A disease allele will give a PCR result and a non-disease allele will not because PCR is designed to amplify the area that corresponds to the mutation on the DNA. If it did not amplify only the target region, there would be too much information and a result would not be possible.



Illustration


Image:PCR_process.gif


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