BME103:T130 Group 3 l2

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(Thermal Cycler Engineering)
Current revision (16:37, 29 November 2012) (view source)
(Protocols)
 
(14 intermediate revisions not shown.)
Line 26: Line 26:
==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 re-design is based upon the [http://openpcr.org Open PCR] system that was originally designed by Josh Perfetto and Tito Jankowski.<br>
'''System Design'''<br>
'''System Design'''<br>
-
[[Image:Reg1.jpg]][[Image:Reg2.jpg]]<br>[[Image:Ch1.jpg]][[Image:Ch2.jpg]]
+
[[Image:G1.jpg]][[Image:G2.jpg]]<br>[[Image:G3.jpg]][[Image:G4.jpg]]
'''Key Features'''<br>
'''Key Features'''<br>
-
The specific feature being focused on is the heating and cooling aspect of the PCR machine. The main alteration of the machine would be to change the material of the: heat sink, heating pad, and  
+
The specific feature being focused on to improve the Open PCR Machine is the heating and cooling aspect of the PCR machine. The main alteration of the machine would be to change the material used on the heat sink and heating pad to copper. This would allow for a much quicker change in temperature while the machine is running. Therefore, the heating and cooling phases of the cycles would be attained faster, thus cutting the time of a full PCR efficiently. The material change from a zinc alloy to copper would make heating and cooling the cycles of the PCR machine much quicker because copper is the most electrically conductive metal.
'''Instructions'''<br>
'''Instructions'''<br>
-
There was only a change in the material of the heat sink and heating pad so the assembly instruction will remain the same as the original design.
+
There was only a change in the material that is used to make the heat sink and heating pad, so the assembly instruction will remain the same as the original design.
<!--- From Week 4 exercise --->
<!--- From Week 4 exercise --->
Line 79: Line 79:
3.) The DNA samples are then heated to ninety-five degrees Celsius (95°C) for one (1) minute  in order to unzip the two single strands. <br>
3.) The DNA samples are then heated to ninety-five degrees Celsius (95°C) for one (1) minute  in order to unzip the two single strands. <br>
4.) They are then cooled to fifty-seven degrees Celsius (57°C) for ten (10) seconds so that the primers can attach to their matching sequences. <br>
4.) They are then cooled to fifty-seven degrees Celsius (57°C) for ten (10) seconds so that the primers can attach to their matching sequences. <br>
-
5.) Finally they are heated back to seventy-two degrees Celsius (72°C) for ten (10) seconds and polymerase extends the DNA strands by attaching the correct free nucleotides in order on the single strands. <br><br>
+
5.) Finally, they are heated back to seventy-two degrees Celsius (72°C) for ten (10) seconds, and polymerase extends the DNA strands by attaching the correct free nucleotides in order on the single strands. <br><br>
<font size=3>'''DNA Measurement Protocol'''</font><br><br>
<font size=3>'''DNA Measurement Protocol'''</font><br><br>
Line 86: Line 86:
<b>'''Fluorimeter Setup'''</b><br>
<b>'''Fluorimeter Setup'''</b><br>
-
1.) The lid was first taken off the the box and one of its sides was unbuttoned in order to create a flap.<br>
+
1.) The lid was first taken off the box and one of its sides was unbuttoned in order to create a flap.<br>
-
2.) The box was the flipped upside down in order to create a dark environment for the camera.<br>
+
2.) The box was then flipped upside down in order to create a dark environment for the camera.<br>
3.) A hydrophobic slide was then inserted into the fluorimeter.<br>
3.) A hydrophobic slide was then inserted into the fluorimeter.<br>
4.) Finally, the camera phone was placed in the stand.
4.) Finally, the camera phone was placed in the stand.
Line 93: Line 93:
<b>Fluorimeter Measurements</b><br>
<b>Fluorimeter Measurements</b><br>
1.) Label transfer pipettes and tubes<br>
1.) Label transfer pipettes and tubes<br>
-
2.) Transfer each sample separately into tube containing 400μl of buffer<Br>
+
2.) Transfer each sample separately into a tube containing 400μl of buffer<Br>
-
3.) Take the specifically labeled tube containing SYBR GREEN 1 and place 2 drops on the first 2 centered drops <br>
+
3.) Take the specifically labeled tube containing SYBR GREEN 1 and place two drops on the first two centered drops <br>
-
4.) Place 2 drops of diluted sample on top of the SYBR GREEN 1 drop<br>
+
4.) Place two drops of diluted sample on top of the SYBR GREEN 1 drop<br>
5.) Align light through drop<br>
5.) Align light through drop<br>
6.) Take pictures using light box<br>
6.) Take pictures using light box<br>
Line 112: Line 112:
9.) Drag circle to the background of the image <br>
9.) Drag circle to the background of the image <br>
10.) Record results <br>
10.) Record results <br>
-
11.) Repeat if necessary
+
11.) Repeat if necessary or needed
<br><br>
<br><br>
Line 123: Line 123:
<!--- 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 single nucleotide polymorphism (SNP), 137852571, that is being examined in this experiment is linked with androgen insensitivity syndrome, and Kennedy spinal and bulbar muscular atrophy. Androgen insensitivity syndrome is when a person who is genetically male (who has one X and one Y chromosome) is resistant to male hormones (called androgens). As a result, the person has some or all of the physical traits of a woman, but the genetic makeup of a man. The mutation on the X chromosome makes the body unable to respond to the hormones that produce a male appearance. Kennedy spinal and bulbar muscular atrophy is a debilitating neurodegenerative disease resulting in muscle cramps and progressive weakness due to degeneration of motor neurons in the brain stem and spinal cord. The SNP is located on the X chromosome and affects the gene AR, the gene is inherited in an x-linked recessive manner therefore only males can be fully affected by the mutation and females are rarely affected. The sequence of this gene is:<br>
+
The single nucleotide polymorphism (SNP), 137852571, that is being examined in this experiment is linked with Androgen Insensitivity Syndrome and Kennedy Spinal and Bulbar Muscular Atrophy. Androgen Insensitivity Syndrome occurs when a person who is genetically male (who has one X and one Y chromosome) is resistant to male hormones (called androgens). As a result, the person has some or all of the physical traits of a female, but the genetic makeup of a male. The mutation on the X chromosome makes the body unable to respond to the hormones that produce a male appearance. Kennedy Spinal and Bulbar Muscular Atrophy is a debilitating neurodegenerative disease resulting in muscle cramps and progressive weakness due to degeneration of motor neurons in the brain stem and spinal cord. The SNP is located on the X chromosome and affects the gene AR, the gene is inherited in an x-linked recessive manner therefore only males can be fully affected by the mutation and females are rarely affected. The sequence of this gene is:<br>
CTTCTCCAGGCTTCCGCAACTTACAC[A/G]TGGACGACCAGATGGCTGTCATTCA<br>
CTTCTCCAGGCTTCCGCAACTTACAC[A/G]TGGACGACCAGATGGCTGTCATTCA<br>
The error in this sequence is represented by [A/G] which means that the normal G base pair has been mutated into an A base pair resulting in an allele that expresses the linked diseases.<br><br>
The error in this sequence is represented by [A/G] which means that the normal G base pair has been mutated into an A base pair resulting in an allele that expresses the linked diseases.<br><br>
-
 
-
Breast Cancer<br>
 
rs137852571 <Br>
rs137852571 <Br>
-
AR –
+
SNP located at:5,255,325 <br>
-
Reverse Primer: <br>
+
-
SNP:5,255,325 <br>
+
Missense GTG→ATG <br>
Missense GTG→ATG <br>
V[Val]→M[Met] <Br>
V[Val]→M[Met] <Br>
Line 155: Line 151:
AGGGGTGGTGGGGAATTACC
AGGGGTGGTGGGGAATTACC
-
'''Illustration'''
+
'''Illustration'''<br>
 +
[[Image:Photogroup3.JPG|600x300px]]
<!--- 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 --->
-
 
+
<b> Bayesian Equation </b> <br><br>
-
 
+
[[Image:58a49e1877dd22125514b93a41037fb1.png|519×48px]] <br>
<!-- ##### 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: Serena KaplanResearch and Development
Name: Serena Kaplan
Research and Development
Name: Gabe McInnisOpen PCR Machine Engineer
Name: Gabe McInnis
Open PCR Machine Engineer
Name: Blake EichlerExperimental Protocol Planner
Name: Blake Eichler
Experimental Protocol Planner
Name: Sierra MorrisExperimental Protocol Planner
Name: Sierra Morris
Experimental Protocol Planner
Name: Zazu MoloiOpen PCR Machine Engineer
Name: Zazu Moloi
Open PCR Machine Engineer
Name: Katelin VaughnResearch and Development
Name: Katelin Vaughn
Research and Development

LAB 2 WRITE-UP

Thermal Cycler Engineering

Our re-design is based upon the Open PCR system that was originally designed by Josh Perfetto and Tito Jankowski.


System Design

Image:G1.jpgImage:G2.jpg
Image:G3.jpgImage:G4.jpg

Key Features
The specific feature being focused on to improve the Open PCR Machine is the heating and cooling aspect of the PCR machine. The main alteration of the machine would be to change the material used on the heat sink and heating pad to copper. This would allow for a much quicker change in temperature while the machine is running. Therefore, the heating and cooling phases of the cycles would be attained faster, thus cutting the time of a full PCR efficiently. The material change from a zinc alloy to copper would make heating and cooling the cycles of the PCR machine much quicker because copper is the most electrically conductive metal.


Instructions
There was only a change in the material that is used to make the heat sink and heating pad, so the assembly instruction will remain the same as the original design.




Protocols

Materials

Supplied in the Kit
PCR Machine
10μM forward primer
10μM reverse primer
GoTaq master mix
Supplied by the User
Template DNA
Distilled Water
Pipets
Eppendorf Tubes

PCR Protocol

1.) Pipet 0.1μL of template DNA, 0.5μL of 10μM forward primer, 0.5μL of 10μM reverse primer, 25.0μL of GoTaq Master Mix, and 23.9μL of dH20 in an Eppendorf tube. All of these items mixed together should create a total volume of 50.0μL.
2.) Up to 16 of these samples are then loaded into the PCR machine
3.) The DNA samples are then heated to ninety-five degrees Celsius (95°C) for one (1) minute in order to unzip the two single strands.
4.) They are then cooled to fifty-seven degrees Celsius (57°C) for ten (10) seconds so that the primers can attach to their matching sequences.
5.) Finally, they are heated back to seventy-two degrees Celsius (72°C) for ten (10) seconds, and polymerase extends the DNA strands by attaching the correct free nucleotides in order on the single strands.

DNA Measurement Protocol



Fluorimeter Setup
1.) The lid was first taken off the box and one of its sides was unbuttoned in order to create a flap.
2.) The box was then flipped upside down in order to create a dark environment for the camera.
3.) A hydrophobic slide was then inserted into the fluorimeter.
4.) Finally, the camera phone was placed in the stand.

Fluorimeter Measurements
1.) Label transfer pipettes and tubes
2.) Transfer each sample separately into a tube containing 400μl of buffer
3.) Take the specifically labeled tube containing SYBR GREEN 1 and place two drops on the first two centered drops
4.) Place two drops of diluted sample on top of the SYBR GREEN 1 drop
5.) Align light through drop
6.) Take pictures using light box
7.) Repeat for each sample.
8.) Run water as BLANK using same procedure

ImageJ Instructions
1.) Open ImageJ
2.) Click ANALYZE tool bar and select SET MEASUREMENTS
3.) Select AREA, MEAN GREY VALUE, and INTEGRATED DENSITY
4.) Upload image to ImageJ
5.) Select IMAGE then COLOR and then SPLIT CHANNELS
6.) Only use green channel
7.) Use OVAL tool and select the entire drop of liquid
8.) Go to ANALYZE and then MEASURE
9.) Drag circle to the background of the image
10.) Record results
11.) Repeat if necessary or needed

Research and Development

Background on Disease Markers

The single nucleotide polymorphism (SNP), 137852571, that is being examined in this experiment is linked with Androgen Insensitivity Syndrome and Kennedy Spinal and Bulbar Muscular Atrophy. Androgen Insensitivity Syndrome occurs when a person who is genetically male (who has one X and one Y chromosome) is resistant to male hormones (called androgens). As a result, the person has some or all of the physical traits of a female, but the genetic makeup of a male. The mutation on the X chromosome makes the body unable to respond to the hormones that produce a male appearance. Kennedy Spinal and Bulbar Muscular Atrophy is a debilitating neurodegenerative disease resulting in muscle cramps and progressive weakness due to degeneration of motor neurons in the brain stem and spinal cord. The SNP is located on the X chromosome and affects the gene AR, the gene is inherited in an x-linked recessive manner therefore only males can be fully affected by the mutation and females are rarely affected. The sequence of this gene is:
CTTCTCCAGGCTTCCGCAACTTACAC[A/G]TGGACGACCAGATGGCTGTCATTCA
The error in this sequence is represented by [A/G] which means that the normal G base pair has been mutated into an A base pair resulting in an allele that expresses the linked diseases.


rs137852571
SNP located at:5,255,325
Missense GTG→ATG
V[Val]→M[Met]
Chromosome X



Primer Design


Normal: G mutates into cancer A
CAACTTACACATGGACGACC

reverse primer:
GTTGAATGTGTACCTGCAGG

Forward primer starts at 5,255,175:
AGGGGTGGTGGGGAATTACC

Illustration

Bayesian Equation

519×48px

Personal tools