BME103:T130 Group 13 l2: Difference between revisions
(9 intermediate revisions by 3 users not shown) | |||
Line 15: | Line 15: | ||
| [[Image:Voltage_main.jpg|100px|thumb|Name: Garrett Repp<br>Open PCR machine engineer]] | | [[Image:Voltage_main.jpg|100px|thumb|Name: Garrett Repp<br>Open PCR machine engineer]] | ||
| [[Image:Joseph_pic.jpeg|100px|thumb|Name: Joseph Rosario<br>Open PCR machine engineer]] | | [[Image:Joseph_pic.jpeg|100px|thumb|Name: Joseph Rosario<br>Open PCR machine engineer]] | ||
| [[Image: | | [[Image:dnaproblems.jpg|100px|100px|thumb|Name: Ujwala Vaka<br>Experimental protocol planner]] | ||
| [[Image:BME103_Group13_emily.jpg|100px|thumb|Name: Emily Herring<br>Experimental protocol planner]] | | [[Image:BME103_Group13_emily.jpg|100px|thumb|Name: Emily Herring<br>Experimental protocol planner]] | ||
| [[Image:BME103_Group13_SIavatar.jpg|100px|thumb|Name: Sudarshan Iyer<br>Research and Development scientist]] | | [[Image:BME103_Group13_SIavatar.jpg|100px|thumb|Name: Sudarshan Iyer<br>Research and Development scientist]] | ||
Line 29: | Line 29: | ||
'''System Design'''<br> | '''System Design'''<br> | ||
[[Image:BME103_13_130_HingedLid_3.JPG|600px|thumb|A-Absent Heated Lid Handle|thumb|A-Absent Heating Lid Handle <br> B- | [[Image:BME103_13_130_HingedLid_3.JPG|600px|thumb|A-Absent Heated Lid Handle|thumb|A-Absent Heating Lid Handle <br> B-Loose Latch|left]] <br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br> | ||
'''Key Features'''<br> | '''Key Features'''<br> | ||
The new design focuses on making the Open PCR easier to use. To accomplish this goal based on the design, the new Open PCR design | The new design focuses on making the Open PCR easier to use. To accomplish this goal based on the design, the new Open PCR design omits the Heated Lid Handle on the old design by making the Heated Lid set in a certain position based on the national standards of the testing vials. Thus, this decreases the error in heating based on a poorly adjusted lid because of this design. The new design also focuses on an improved latching system. This latch previously had difficulties opening and closing because of the tight fit. This has been ameliorated by making the latch loose in order to open and close more easily. | ||
'''Instructions'''<br> | '''Instructions'''<br> | ||
To access the 16 tube rack, gently pull up on the lip of the lid (located on the same side as the latch). After loading or unloading the tube rack, close the lid by slowly, yet firmly pushing down on the top of the lid until it clicks into place. Make sure to double check to see if it | To access the 16 tube rack, gently pull up on the lip of the lid (located on the same side as the latch). After loading or unloading the tube rack, close the lid by slowly, yet firmly pushing down on the top of the lid until it clicks into place. Make sure to double check to see if it closes all the way by slightly pulling up on the lid without opening it, to see if the latch prevents it from opening. Now you are free to run the test. The rest of the machine should work exactly as it did before the moderation. | ||
<!--- From Week 4 exercise ---> | <!--- From Week 4 exercise ---> | ||
Line 64: | Line 64: | ||
{| border="1" cellpadding="5" cellspacing="0" align="center" | {| border="1" cellpadding="5" cellspacing="0" align="center" | ||
! scope="col" | Supplied in Kit | ! scope="col" | Supplied in Kit | ||
|- | |- | ||
|PCR Master Mix | |PCR Master Mix | ||
|- | |- | ||
|PCR Machine | |PCR Machine | ||
|- | |- | ||
|SYBR Green I | |SYBR Green I | ||
|- | |- | ||
|Calf Thymus DNA Solution | |Calf Thymus DNA Solution | ||
|- | |- | ||
|Fluorimeter Kit | |Fluorimeter Kit | ||
|- | |- | ||
|Mat with measurements | |Mat with measurements | ||
|} | |} | ||
Line 94: | Line 87: | ||
{| border="1" cellpadding="5" cellspacing="0" align="center" | {| border="1" cellpadding="5" cellspacing="0" align="center" | ||
! scope="col" | Supplied by User | ! scope="col" | Supplied by User | ||
|- | |- | ||
|Smartphone | |Smartphone | ||
|- | |- | ||
|Pipettes | |Pipettes | ||
|- | |- | ||
|Eppendorf Tubes | |Eppendorf Tubes | ||
|- | |- | ||
|DNA Samples | |DNA Samples | ||
|- | |- | ||
|Water | |Water | ||
|} | |} | ||
Line 143: | Line 130: | ||
12) Repeat steps 5-10 four more times in 5 different positions. <br> | 12) Repeat steps 5-10 four more times in 5 different positions. <br> | ||
After assembling the fluorimeter, you can now determine if you've amplified the targeted DNA in your PCR experiment. Using the Fluorimeter and the smartphone app, you can calculate the relative amount of DNA through fluorescence, which is generated by excitation and emission wavelengths. In order to detect fluorescence when dsDNA is present, you'll be using SYBR Green I because it's more safer compared to other dyes. With that being said, gloves must be worn when handling any liquid containing SYBR Green I. The fluorimeter itself is a very simple machine because it uses optical caustic, a special type of optics that completely removes the need for lasers, mirrors, or lenses. Also the flourimeter is battery-powered, lightweight and portable; this allows every student to have one of these at their lab table. Following the steps below, you can easily learn how to dye your amplified DNA. | After assembling the fluorimeter, you can now determine if you've amplified the targeted DNA in your PCR experiment. Using the Fluorimeter and the smartphone app, you can calculate the relative amount of DNA through fluorescence, which is generated by excitation and emission wavelengths. In order to detect fluorescence when dsDNA is present, you'll be using SYBR Green I because it's more safer compared to other dyes. With that being said, gloves must be worn when handling any liquid containing SYBR Green I. The fluorimeter itself is a very simple machine because it uses optical caustic, a special type of optics that completely removes the need for lasers, mirrors, or lenses. Also the flourimeter is battery-powered, lightweight and portable; this allows every student to have one of these at their lab table. Following the steps below, you can easily learn how to dye your amplified DNA. <br> | ||
1)On your lab table, you'll find eight samples from the Open PCR, 1 DNA sample(calf thymus standard at 2 micrograms/mL), and water from the scintillation vial (white cap) to analyze. <br> | 1)On your lab table, you'll find eight samples from the Open PCR, 1 DNA sample(calf thymus standard at 2 micrograms/mL), and water from the scintillation vial (white cap) to analyze. <br> | ||
2)With a permanent marker, label your Eppendorf tubes and number your pipettes (on the bulb part) so that no cross-contamination will occurs. At the end, you should have 10 Eppendorf tubes and 10 pipettes clearly labeled (see Table 3). REMINDER: Use only 1 transfer pipette per sample!!! <br> | 2)With a permanent marker, label your Eppendorf tubes and number your pipettes (on the bulb part) so that no cross-contamination will occurs. At the end, you should have 10 Eppendorf tubes and 10 pipettes clearly labeled (see Table 3). REMINDER: Use only 1 transfer pipette per sample!!! <br> | ||
Line 176: | Line 164: | ||
'''Primer Design''' | '''Primer Design''' | ||
<br><br>Since this SNP lies on the forward strand, the primer used to facilitate a DNA signal of the mutated DNA will include a forward primer containing the mutation and a reverse primer 200 base pairs to the right of the mutation, with each primer being 20 base pairs long. | <br><br>Since this SNP lies on the forward strand, the primer used to facilitate a DNA signal of the mutated DNA will include a forward primer containing the mutation and a reverse primer 200 base pairs to the right of the mutation, with each primer being 20 base pairs long. | ||
<br>Forward Primer (5' to 3'): | <br><br>Forward Primer (5' to 3'): [[Image:BME103_Group13_SIForwardprimer.png]] | ||
<br>Reverse Primer (5' to 3'): | <br>Reverse Primer (5' to 3'): [[Image:BME103_Group13_SIReverseprimer.png]] | ||
<br><br> A disease allele will produce a PCR product while a non-disease will not produce a PCR product due to the ability of the forward primer, which contains the mutated T, to bind to the reverse strand. If the reverse strand has a corresponding A at the position opposite the mutated T then the primer will bind. If the reverse strand has a C instead (normal), the forward primer will not bind to the reverse strand. This is illustrated in the figure below. | |||
<!--- 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. ---> | ||
'''Illustration''' | '''Illustration''' | ||
<br><br>[[Image:BME103_Group13_SIPrimer.png]] | |||
'''Bayes' Rule: Test of Reliability''' | |||
<br><br>A nucleic acid test is only as useful as its level of reliability. If the test is not reliable and used, people who have a mutated T will be told they have or will have hipohydrotic ectodermal dysplasia when they actually do not. Bayes' Rule provides an equation that can be used to determine the probability that a person who has a mutated T will have hipohydrotic ectodermal dysplasia. The equation with the necessary variables is displayed below. | |||
<br>[[Image:BME103_Group13_SIBRule.png]] | |||
<!--- 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 13:26, 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
ProtocolsMaterials
PCR Protocol Adjusting smartphone settings:
DNA Measurement Protocol After assembling the fluorimeter, you can now determine if you've amplified the targeted DNA in your PCR experiment. Using the Fluorimeter and the smartphone app, you can calculate the relative amount of DNA through fluorescence, which is generated by excitation and emission wavelengths. In order to detect fluorescence when dsDNA is present, you'll be using SYBR Green I because it's more safer compared to other dyes. With that being said, gloves must be worn when handling any liquid containing SYBR Green I. The fluorimeter itself is a very simple machine because it uses optical caustic, a special type of optics that completely removes the need for lasers, mirrors, or lenses. Also the flourimeter is battery-powered, lightweight and portable; this allows every student to have one of these at their lab table. Following the steps below, you can easily learn how to dye your amplified DNA. 1)On your lab table, you'll find eight samples from the Open PCR, 1 DNA sample(calf thymus standard at 2 micrograms/mL), and water from the scintillation vial (white cap) to analyze. Uploading Pictures and Analysis Using ImageJ
Research and DevelopmentBackground on Disease Markers
Bayes' Rule: Test of Reliability
|
|||||||||||||||||||