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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'''DNA Measurement Protocol''' <br> | '''DNA Measurement Protocol''' <br> | ||
'''Flourimeter Procedure'''<br> | |||
1. Turn on the excitation light using the switch for the Blue LED. <br> | |||
2. Place your smart phone on the cradle at a right angle from the slide. <br> | |||
3. Turn on the camera setting on the smartphone. Turn off the flash and set the ISO to 800 or higher and increase the exposure to maximum. You should also turn off the autofocus, if possible, and make sure that you can take an image where the drop on the slide will be in focus. <br> | |||
4. Adjust the distance between the smartphone on its cradle and the first two rows of the slide so that it is as close as you can get without having a blurry image. <br> | |||
5. The pipette should be filled with liquid only to the bottom of the black line. Then, carefully place two drops of water in the middle of the first two rows of the slide using the plastic pipette. Then add two more drops. The drop should then be pinned and look like a beach ball. It should be between 130-160 μL. <br> | |||
6. Align the drop by moving the slide so that the blue LED light is focused by the drop to the middle of the black fiber optic fitting on the other side of the drop (you will see that it has a small opening that is used for spectral measurement). <br> | |||
7. Cover the fluorimeter with the light box, but make sure you can access your smartphone to take the image. The light box should be used to remove as much stray light as possible, but do not worry if you have some light. <br> | |||
8. Take three images of the drop of water. Do not move your smartphone. <br> | |||
9. Remove the box and be careful not to move your smartphone. If you want to adjust for any movement, use the ruler provided to measure the distance so that you can return to that location. You can also use ImageJ to compensate for moving the camera, but it makes the analysis more complicated. <br> | |||
10. Use a clean plastic pipette to remove the water drop from the surface. <br> | |||
11. Push the slide in so that you are now in the next set of two holes. <br> | |||
12. Repeat steps 5-10 four more times so that you have now imaged all 5 positions on the slide. <br> | |||
13. Record the type of smartphone you used, the distance from the base of smartphone cradle to measurement device, and attach one image for each position of the drop. <br> | |||
'''Image J Procedure'''<br> | '''Image J Procedure'''<br> | ||
1. Search Image J in Google and then download Image J <br> | 1. Search Image J in Google and then download Image J <br> | ||
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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>. |