BME103:T930 Group 1 l2: Difference between revisions
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| PCR Machine || 1 | | PCR Machine || 1 | ||
|- | |- | ||
| | | Fluorimeter || 1 | ||
|- | |- | ||
| | | Black Box || 1 | ||
|- | |- | ||
| | | Positive Control DNA Sample|| 100 μL | ||
|- | |- | ||
| | | Negative Control DNA Sample || 100 μL | ||
|- | |- | ||
| | | 10 μM Forward Primer || 10 μL | ||
|- | |- | ||
| | | 10 μM Reverse Primer || 10 μL | ||
|- | |- | ||
| PCR | | GoTaq Colorless Master Mix || 400 μL | ||
|- | |||
| Eppendorf Tubes || 8 | |||
|- | |||
| Test Tubes || 8 | |||
|- | |||
| Pipettes || 10 | |||
|- | |||
| Hydrophobic Glass Slides || 5 | |||
|- | |||
| Tris Buffer (0.025% SYBR Green) || 100 μL | |||
|} | |||
<br> | |||
{| {{table}} | |||
|- style="background:#f0f0f0;" | |||
| '''Supplied by User''' || '''Amount''' | |||
|- | |||
| DNA Samples || 200 μL | |||
|- | |||
| Open PCR Software || 1 | |||
|- | |||
| ImageJ Software || 1 | |||
|- | |||
| Smartphone || 1 | |||
|- | |||
| Smartphone Cradle || 1 | |||
|- | |||
| Distilled Water || 50 mL | |||
|} | |} | ||
<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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<!--- Bonus: explain how Bayesian statistics can be used to assess the reliability of your team's method. Just write the equation using variables that are relevant to your team's new test. You do not need actual numbers ---> | <!--- Bonus: explain how Bayesian statistics can be used to assess the reliability of your team's method. Just write the equation using variables that are relevant to your team's new test. You do not need actual numbers ---> | ||
'''Bayes Theorem''' <br> | |||
P(A/B)= (P(B/A)P(A))/(P(B))<br> | |||
Bayes Theorem is used to determine if the amount of accurate results outweigh the false positives/false negatives that the PCR machine gives. In other words, Bayes Theorem shows either that the PCR machine gives far more accurate results than false positives/negatives (and is therefore worth building and marketing) or that the PCR machine gives fewer accurate results than false positives/negatives (and is therefore not worth building and marketing). <br> | |||
'''Background on Disease Markers''' | '''Background on Disease Markers''' | ||
Diabetic insipidus is a kidney disease in which the kidneys are unable to conserve water[http://www.ncbi.nlm.nih.gov/pubmedhealth/PMH0001415/]. This is controlled by antidiuretic hormone (or vasopressin), and in diabetic insipidus there is a lack of this hormone. It can be caused by damage to the hypothalamus or pituitary gland. This hormone is produced in the hypothalamus and is released from the pituitary gland. The SNP rs121964890[http://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?rs=121964890] can cause DI.[http://omim.org/entry/603933] It is located on the 20th chromosome and has the allele change TCC ⇒ TTC which is the residue change S [Ser] ⇒ F [Phe]. | |||
<!--- 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) ---> | ||
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'''Primer Design''' | '''Primer Design''' | ||
3' GAAGACCGGC[C/T]TGACCGTCCC 5' | |||
5' GGGACGGTGA[G/A]GCCGGTCTTC 3' | |||
TCC ⇒ TTC | |||
S [Ser] ⇒ F [Phe] | |||
<!--- 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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'''Illustration''' | '''Illustration''' | ||
[[Image:PrimerAmplification.gif]] | |||
<!--- 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 10:42, 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
Instructions
ProtocolsMaterials
1. You will need a PCR machine and computer. 2. Download the PCR software onto the computer. 3.Thaw the GoTaq Colorless Master Mix at room temperature. Vortex the Master Mix, then spin it briefly in a microcentrifuge to collect the material at the bottom of the tube.
5. If using a cycler without a heated lid, overlay the reaction mix with 1-2 drops of mineral oil to prevent evaporation during thermal cycling. Centrifuge the reaction mix in a microcentrifuge for 5 seconds. 6. Place the reactions in a thermal cycler that has been preheated to 95 degrees Celsius. Perform PCR. What Occurs In the PCR Machine 2. Annealing: perform the reaction about 5 degrees Celsius below the calculated melting temperature of the primers and increasing the temperature in increments of 1°C to the annealing temperature; this should occur anywhere between 30 seconds and 1 minute. 3. Extension: performed between 72-74 degrees Celsius, extension allows 1 minute for every 1 kb of DNA to be amplified; the suggested time for extension is 5 minutes. 4. Refrigeration: refrigerate the tubes at 4 degrees Celsius for several hours; this will minimize the opportunity for DNA polymerase to continue to be active at higher temperatures. 5. Cycle Number: the optimal amplification is 25-30 cycles, but up to 40 may be performed.
1. Turn on the excitation light using the switch for the Blue LED. Image J Procedure Research and DevelopmentBayes Theorem P(A/B)= (P(B/A)P(A))/(P(B)) Bayes Theorem is used to determine if the amount of accurate results outweigh the false positives/false negatives that the PCR machine gives. In other words, Bayes Theorem shows either that the PCR machine gives far more accurate results than false positives/negatives (and is therefore worth building and marketing) or that the PCR machine gives fewer accurate results than false positives/negatives (and is therefore not worth building and marketing).
Diabetic insipidus is a kidney disease in which the kidneys are unable to conserve water[1]. This is controlled by antidiuretic hormone (or vasopressin), and in diabetic insipidus there is a lack of this hormone. It can be caused by damage to the hypothalamus or pituitary gland. This hormone is produced in the hypothalamus and is released from the pituitary gland. The SNP rs121964890[2] can cause DI.[3] It is located on the 20th chromosome and has the allele change TCC ⇒ TTC which is the residue change S [Ser] ⇒ F [Phe].
3' GAAGACCGGC[C/T]TGACCGTCCC 5' 5' GGGACGGTGA[G/A]GCCGGTCTTC 3' TCC ⇒ TTC S [Ser] ⇒ F [Phe]
References"GoTaq® Colorless Master Mix (M714) Product Information." GoTaq® Colorless Master Mix Protocol. Promega, 2012. Web. 15 Nov. 2012. <http://www.promega.com/resources/protocols/product-information-sheets/g/gotaq-colorless-master-mix-m714-protocol/>. Hunt, Margaret. "Real Time PCR Tutorial." Real Time PCR Tutorial. University of South Carolina, 10 July 2010. Web. 15 Nov. 2012. <http://pathmicro.med.sc.edu/pcr/realtime-home.htm>. |