BME103 s2013:T900 Group9: Difference between revisions
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| [[Image: | | [[Image:BME103 Group9ColeyWhite Assembly.jpg|100px|thumb|Name: Coley White<br>Role(s): Protocol Planner]] | ||
| [[Image: | | [[Image:BME103 Group3 AimenVanood.jpg|100px|thumb|Name: Aimen Vanood<br>Role(s): Research and development]] | ||
| [[Image:Nordy.gif|100px|thumb|Name: Brady Falk<br>Role(s): Machine testing]] | | [[Image:Nordy.gif|100px|thumb|Name: Brady Falk<br>Role(s): Machine testing]] | ||
| [[Image:BME103student.jpg|100px|thumb|Name: student<br>Role(s)]] | | [[Image:BME103student.jpg|100px|thumb|Name: student<br>Role(s)]] | ||
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==Initial Machine Testing== | ==Initial Machine Testing== | ||
[[Image:PCR.jpg|400px|]]<br> | |||
'''The Original Design'''<br> | '''The Original Design'''<br> | ||
A Polymerase Chain Reaction (PCR) Machine, as shown in the above image, is a machine that is meant to produce large quantities of specific Deoxyribose Nucleic Acid (DNA) sequences. The method used is by completing several heating and cooling cycles to unzip DNA strands and isolate the desired DNA strands.<br> | |||
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'''Thermal Cycler Program'''<br> | '''Thermal Cycler Program'''<br> | ||
<p>'''Heated Lid:''' 110°C</p> | |||
<p>'''Initial Step:''' temp: 95°C time: 180 sec </p> | |||
<p>'''Number of Cycles:''' 35 </p> | |||
<p>'''Denaturing:''' temp: 95°C time: 30 sec</p> | |||
<p>'''Annealing:''' temp: 57°C time: 30 sec</p> | |||
<p>'''Extending:''' temp: 72°C time: 30 sec</p> | |||
<p>'''Final Hold:''' temp: 4°C</p> | |||
After the thermal cycler program is set up, we then proceeded with the experiment by saving the settings and selecting the "Plug in OpenPCR to Start" option. | |||
'''DNA Sample Set-up'''<br> | '''DNA Sample Set-up'''<br> | ||
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{| {{table}} | {| {{table}} | ||
|- | |- | ||
| <p>Positive control:</p> <p>cancer DNA template</p> Tube label: | | <p>Positive control:</p> <p>cancer DNA template</p> Tube label: '''+''' || <p>Patient 1</p> <p> ID: '''10840'''</p> <p>Replicate 1</p> <p> Tube label '''A1'''</p> || <p>Patient 1</p> <p> ID: '''10840'''</p> <p>Replicate 2</p> <p> Tube label '''A2'''</p> || <p>Patient 1</p> <p> ID: ['''10840'''</p> <p>Replicate 3</p> <p> Tube label '''A3'''</p> | ||
|- | |- | ||
| <p>Negative control:</p> <p>cancer DNA template</p> Tube label: | | <p>Negative control:</p> <p>cancer DNA template</p> Tube label: '''-''' || <p>Patient 2</p> <p> ID: '''12675'''</p> <p>Replicate 1</p> <p> Tube label '''B1'''</p> || <p>Patient 2</p> <p> ID: '''12675'''</p> <p>Replicate 2</p> <p> Tube label '''B2'''</p> || <p>Patient 2</p> <p> ID: '''12675'''</p> <p>Replicate 3</p> <p> Tube label '''B3''' | ||
|} | |} | ||
'''DNA Sample Set-up Procedure''' | '''DNA Sample Set-up Procedure''' | ||
# Step 1: First, we | # Step 1: First, we collected all the materials and correctly identified the eight testing tubes, with a marker, according to the corresponding ID, patient, and replication. | ||
# Step 2: Then | # Step 2: Then we transferred 50 μL of the given PCR reaction mix into the 8 tubes using a micropipette. It is imperative that new tips are used each time and then properly disposed of to avoid contamination. Also the container of the tips must remain shut when it is not in used so that airborne contamination is avoided as well. | ||
# Step 3: Next, | # Step 3: Next, we transferred 50 μL of the DNA/ primer mix into the 8 tubes, but unlike the reaction mix, the DNA must be carefully transferred into the correct tubes. The first four tubes would have one tube that represented the positive control and the next three would contain the DNA of patient 1. For the remaining tubes, one will be used as the negative control and the rest of the three tubes will contain the DNA of patient 2. | ||
# Step 4: Then, the mixtures will need to be placed into the machine, which must be programmed to the correct temperatures and cycles. | # Step 4: Then, the mixtures will need to be placed into the machine, which must be programmed to the correct temperatures and cycles. | ||
'''PCR Reaction Mix''' | '''PCR Reaction Mix''' | ||
* | The mix is composed of:</p> | ||
<p>* Taq DNA polymerase</p> | |||
<p>* MgCl2</p> | |||
<p>* dNTP’s.</p> | |||
'''DNA/ primer mix''' | '''DNA/ primer mix''' | ||
* | <p>The DNA/ primer mix is composed of:</p> | ||
<p>* Either one of the DNA of patient 1 or patient 2. </p> | |||
<p>* A forward and reverse primer, which will be the same and present in all tubes. </p> | |||
Latest revision as of 11:19, 2 April 2013
BME 103 Spring 2013 | Home People Lab Write-Up 1 Lab Write-Up 2 Lab Write-Up 3 Course Logistics For Instructors Photos Wiki Editing Help | ||||||||||||||
OUR TEAMLAB 1 WRITE-UPInitial Machine TestingThe Original Design
When we unplugged the Liquid Crystal Display from the open PCR circuit board, the machine no longer displayed onything on the LCD screen. When we unplugged the white wire that connects the circuit board to 16-tube PCR block, the machine would not produce any heat through the 16-tube PCR block. Anything connected to the circuit board, such as the LCD display or the 16-tube PCR block, will not run when the connection is disrupted.
The first test was run on March 5, 2013 using Machine 13. The sample of DNA was put into the PCR machine and all of the results came out as expected. There were no problems while testing.
ProtocolsThermal Cycler Program Heated Lid: 110°C Initial Step: temp: 95°C time: 180 sec Number of Cycles: 35 Denaturing: temp: 95°C time: 30 sec Annealing: temp: 57°C time: 30 sec Extending: temp: 72°C time: 30 sec Final Hold: temp: 4°C After the thermal cycler program is set up, we then proceeded with the experiment by saving the settings and selecting the "Plug in OpenPCR to Start" option. DNA Sample Set-up
DNA Sample Set-up Procedure
PCR Reaction Mix The mix is composed of:* Taq DNA polymerase * MgCl2 * dNTP’s. DNA/ primer mix The DNA/ primer mix is composed of: * Either one of the DNA of patient 1 or patient 2. * A forward and reverse primer, which will be the same and present in all tubes.
Research and DevelopmentSpecific Cancer Marker Detection - The Underlying Technology (Add a write-up of the information discussed in Week 3's class) (BONUS points: Use a program like Powerpoint, Word, Illustrator, Microsoft Paint, etc. to illustrate how primers bind to the cancer DNA template, and how Taq polymerases amplify the DNA. Screen-captures from the OpenPCR tutorial might be useful. Be sure to credit the source if you borrow images.)
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