BME103:T130 Group 2 l2: Difference between revisions
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| [[Image:BME103_group2_12.jpg|100px|thumb|Name: Charul Singh<br>Experimental Protocol Planner]] | | [[Image:BME103_group2_12.jpg|100px|thumb|Name: Charul Singh<br>Experimental Protocol Planner]] | ||
| [[Image:BME103_Group2_AlexHoffmann.jpg|100px|thumb|Name: Alex Hoffmann<br>Experimental Protocol Planner]] | | [[Image:BME103_Group2_AlexHoffmann.jpg|100px|thumb|Name: Alex Hoffmann<br>Experimental Protocol Planner]] | ||
| [[Image: | | [[Image:AJ101.jpg|100px|thumb|Name: AJ Ciferno<br>PCR Machine Engineer]] | ||
| [[Image:Dog.jpeg|100px|thumb|Name: Haley Gjertsen<br>Research and Development Specialist]] | | [[Image:Dog.jpeg|100px|thumb|Name: Haley Gjertsen<br>Research and Development Specialist]] | ||
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'''System Design'''<br> | '''System Design'''<br> | ||
[[Image:Lid ISO.JPG]] | [[Image:Lid ISO.JPG]]<br> | ||
Our new design for the PCR lid includes a see through glass lid along with a door opening like an oven's to slide the samples into place. The new lid will be double layered in order to fit twice the amount of samples. Therefore, there will be a shelf inside to allow the sliding of samples on the top and on the bottom when the door is open. We redesigned to be glass in order to allow better viewing of the samples. The machine will involve the same tightening screw technique of the previous PCR design. Before the glass redesign, we were unable to visually see how tight the lid was on the samples. This brought up the possibility of us tightening the lid too tight, thereby squishing the samples. Moreover, this led to the DNA sample tubes melting. Now that we have the transparent glass surface, we are able to see how tight the lid is, preventing us from harming the samples. | |||
'''Key Features'''<br> | '''Key Features'''<br> | ||
A. | A. Now designed to be on the side, serves as the tightening knob as in the previous design | ||
B. Glass oven like | B. Glass oven-like door on the side | ||
C. | C. Double layered to fit twice the amount of samples (32 samples)<br> | ||
Areas marked by "*" indicate sections that will now be made from glass and therefore, transparent allowing for observation. | |||
New area for samples opens like an oven where the samples can be slid in. It will hold 32 samples instead of its original, 16. The sample trays will be placed one on top of an other when slid in onto the shelf. The glass is for seeing how tightly closed the oven lid is screwed on. | |||
'''Instructions'''<br> | '''Instructions'''<br> | ||
1. Unscrew glass "oven-like door"<br> | |||
2. Slide 16 samples onto the top shelf and 16 samples into the bottom shelf<br> | |||
3. Close door<br> | |||
4. Screw the door tight but not until it touches the sides of the sample ependorf tube<br> | |||
5. Run PCR as original<br><br> | |||
'''Benefits'''<br> | |||
With the new design the PCR machine can now run more samples at a time. Therefore, the efficiency of the system is increased. Also, there will now be an absolute way of determining whether the door is too tight on the tubes. This allows us to be positive that the PCR machine will perform the best test on the DNA samples. | |||
<!--- From Week 4 exercise ---> | <!--- From Week 4 exercise ---> | ||
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---> | ---> | ||
'''Materials''' | '''Materials'''<br> | ||
{| | |||
| align="center" style="background:#f0f0f0;"|'''Supplied in Kit''' | |||
| align="center" style="background:#f0f0f0;"|'''Amount''' | |||
|- | |||
| PCR machine with new glass top||1: $400 to $600 | |||
|- | |||
| Fluorometer: (LED light glass stand)||1: $200.00 | |||
|- | |||
| Connection cord for Computer to PCR||1: $20.00 | |||
|- | |||
| Smartphone Stand||1: $5.00 | |||
|- | |||
| Box||1: $5.00 | |||
|- | |||
| Disk for ImageJ program||1: $0.50 | |||
|- | |||
| Shelf and New glass lid||1: $100.00 | |||
|- | |||
| | |||
|}<br> | |||
{| | |||
| align="center" style="background:#f0f0f0;"|'''Supplied by User''' | |||
| align="center" style="background:#f0f0f0;"|'''Amount''' | |||
|- | |||
| Computer ||1: $1000.00 | |||
|- | |||
| Pipettes||16: $0.25each | |||
|- | |||
| Glass Slides||2: $1.00each | |||
|- | |||
| Tubes||32: $4.00 | |||
|- | |||
| Smartphone||1: $200.00 | |||
|- | |||
| Water||varies | |||
|- | |||
| DNA samples||varies | |||
|- | |||
| | |||
|}<br> | |||
{| | |||
| align="center" style="background:#f0f0f0;"|'''DNA Reagent Materials''' | |||
| align="center" style="background:#f0f0f0;"|'''Amount''' | |||
|- | |||
| Patient's Template DNA||0.2 μL | |||
|- | |||
| GoTaq master mix||50.0 μL | |||
|- | |||
| dH2O||47.8 μL | |||
|- | |||
| 10 μM Reverse Primer||1.0 μL | |||
|- | |||
| 10 μM Forward Primer||1.0 μL | |||
|- | |||
| Total||100 μL | |||
|- | |||
| | |||
|}<br> | |||
<!--- Place your two tables "Supplied in the kit" and "Supplied by User" here ---> | <!--- Place your two tables "Supplied in the kit" and "Supplied by User" here ---> | ||
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'''Background on Disease Markers'''<br> | '''Background on Disease Markers'''<br> | ||
'''Transient Myeloproliferative Disorder of Down Syndrome''' is a combination of the diseases leukemia, down syndrome and acute myeloid. It is extremely rare and takes the life of a patient in the very early months of life. It is associated with the SNP (single nucleotide polymorphism) 121912500 on chromosome 21.<br> | '''Transient Myeloproliferative Disorder of Down Syndrome''' is a combination of the diseases leukemia, down syndrome and acute myeloid. It is extremely rare and takes the life of a patient in the very early months of life. It is associated with the SNP (single nucleotide polymorphism) 121912500 on chromosome 21.<br> | ||
http://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?rs=121912500<br> | http://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?rs=121912500<br><br> | ||
Our PCR machine can test for multiple DNA mutations such as cancer, like in previous experiments, and now Transient Myeloproliferative Disorder of Down Syndrome.<br><br> | |||
'''Primer Design'''<br> | '''Primer Design'''<br> | ||
'''Transient Myeloproliferative Diorder of Down Syndrome:''' The forward primer of this disease is ACCGGCTG'''G'''TGGGCCCGCTG. The reverse primer that would correctly match the forward primer and not code for a diseased patient would be TGGCCGAC'''C'''ACCCGGGCGAC. The diseased allele and diseased patient will have a reverse primer TGGCCGAC'''A'''ACCCGGGCGAC. Therefore, a patient that contains the disease has an allele mutation within the pairing of G to C, it is instead G to A. This will give a positive on the PCR machine because it becomes single stranded due to the fact it cannot replicate properly. This, when we apply the SYBR green it will attach to only the single primers and the diseased patients will glow green. The replicated, normal strands, will appear clear when the SYBR green | '''Transient Myeloproliferative Diorder of Down Syndrome:''' The forward primer of this disease is ACCGGCTG'''G'''TGGGCCCGCTG. The reverse primer that would correctly match the forward primer and not code for a diseased patient would be TGGCCGAC'''C'''ACCCGGGCGAC. The diseased allele and diseased patient will have a reverse primer TGGCCGAC'''A'''ACCCGGGCGAC. Therefore, a patient that contains the disease has an allele mutation within the pairing of G to C, it is instead G to A. This will give a positive on the PCR machine because it becomes single stranded due to the fact it cannot replicate properly. This, when we apply the SYBR green to it, will attach to only the single primers and the diseased patients will glow green. The replicated, normal strands, will appear clear when the SYBR green is added because it will not be able to attach to the replicated strands. | ||
<!--- 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. ---> | ||
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'''5. The process repeats as the PCR machine heats up to 95 degrees.'''<br> | '''5. The process repeats as the PCR machine heats up to 95 degrees.'''<br> | ||
<br><br> | <br><br> | ||
(Photo Credit: The images used here were borrowed from OpenPCR.) | (Photo Credit: The images used here were borrowed from OpenPCR.)<br> | ||
'''Bonus:'''<br> | |||
Bayesian statistics allows you to analyze and use all available data and can help you under the limitations of diagnostic tests. In our case, it can be used to test a sample of patients for this disease. From the tests we can see the false positive rate, sensitivity the test has to the disease, and prevalence to better inform the public on the statistics of the disease within the population and the subject to failure the test has so the doctors can run more if necessary. <br> | |||
D=disease positive<br> | |||
T=positive test<br> | |||
p=probability<br> | |||
p(DIT)=[p(TID)*p(D)]/[(p(TID)*p(D))+(p(TI~D)*p(~D))] | |||
<!--- 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 13:46, 29 November 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.
Key Features A. Now designed to be on the side, serves as the tightening knob as in the previous design B. Glass oven-like door on the side C. Double layered to fit twice the amount of samples (32 samples) Areas marked by "*" indicate sections that will now be made from glass and therefore, transparent allowing for observation. New area for samples opens like an oven where the samples can be slid in. It will hold 32 samples instead of its original, 16. The sample trays will be placed one on top of an other when slid in onto the shelf. The glass is for seeing how tightly closed the oven lid is screwed on. Instructions
ProtocolsMaterials
Setting up the smart phone camera
Set the smart phone camera menu to the following:
1. Inactivate the flash.
DNA Measurement Protocol
Research and DevelopmentBackground on Disease Markers Primer Design
Illustration
|