BME100 f2014:Group27 L4: Difference between revisions
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| [[Image:BME103student.jpg|100px|thumb|Jonathan Almendras]] | | [[Image:BME103student.jpg|100px|thumb|Jonathan Almendras]] | ||
| [[Image:BME103student.jpg|100px|thumb|Patrick Conely]] | | [[Image:BME103student.jpg|100px|thumb|Patrick Conely]] | ||
| [[Image: | | [[Image:Krystal Corette's Face.jpg|100px|thumb|Krystal Corrette]] | ||
| [[Image: | | [[Image:Aaron Dodell's Face.jpg|100px|thumb|Aaron Dodell]] | ||
| [[Image:BME103student.jpg|100px|thumb|Adam Samuel]] | | [[Image:BME103student.jpg|100px|thumb|Adam Samuel]] | ||
| [[Image: | | [[Image:Yakut Umar's Face.JPG|100px|thumb|Yakut Umar]] | ||
|} | |} | ||
=Lab 4: PCR DNA Amplification= | =Lab 4: PCR DNA Amplification= | ||
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'''Purpose/Goal:''' To successfully amplify a section of DNA containing a disease marker in order to accurately diagnose patients. | '''Purpose/Goal:''' To successfully amplify a section of DNA containing a disease marker in order to accurately diagnose patients. | ||
'''Materials:''' | '''Materials:''' | ||
: a. Lab coat and disposable gloves | |||
: b. PCR reaction mix, 8 tubes, 50 uL each: Mix containts Taq DNA polymerase, MgCl2, and dNPT's | |||
: c. DNA/primer mix, 8 tubes, 50 uL each: Each mix contains a different template DNA. All tubes have the same forward primer and reverse primer. | |||
: d. A strip of empty PCR tubes | |||
: e. Disposable pipette tips: only use each one once. Never re-use disposable pipette tips or samples will be cross-contaminated. | |||
: f. Cup for discarded tips | |||
: g. Micropipettor | |||
: h. OpenPCR machine: shared by two groups | |||
'''PCR Reaction Sample List:''' | '''PCR Reaction Sample List:'''<br> | ||
{| {{table}} | |||
|- | |||
| <b>Tube Label</b> || <b>PCR Reaction Sample</b> || <b>Patient ID</b> | |||
|- | |||
| G27 P || Positive control || none | |||
|- | |||
| G27 N || Negative control || none | |||
|- | |||
| G27 1-1 || Patient 1, replicate 1 || 32955 | |||
|- | |||
| G27 1-2 || Patient 1, replicate 2 || 32955 | |||
|- | |||
| G27 1-3 || Patient 1, replicate 3 || 32955 | |||
|- | |||
| G27 2-1 || Patient 2, replicate 1 || 33559 | |||
|- | |||
| G27 2-2 || Patient 2, replicate 2 || 33559 | |||
|- | |||
| G27 2-3 || Patient 2, replicate 3 || 33559 | |||
|} | |||
'''DNA Sample Set-Up Procedure'''<br> | |||
: 1. Without opening the tubes, obtain patient samples along with the positive and negative controls. Each set has its own patient ID number, and there should be 3 copies of each patient sample. | |||
: 2. Create the report by replacing the '#' with your group's number (27) and then record the Patient IDs in your table/list. Remember to not actually write anything on the tubes.<br> | |||
'''OpenPCR Program'''<br> | |||
[[Image:PCR Diagram Group 27.jpg|thumb|center|Basic methodology behind PCR practice. Separate strands chemically, then synthesize complementary DNA strands from the original templates with polymerases. Repeat ad nauseum.]] | |||
Run the heating and cooling program on the thermal cycler.<br> | |||
: 1. Heating Lid at 100°C <br> | |||
: 2. Keep at 95°C for about 2 minutes <br> | |||
: 3. Run 35 cycles, then denature at 95°C for 30 seconds. | |||
: 4. Anneal at 57°C for 30 seconds, then extend at 72°C for 30 seconds. | |||
: 5. Finally, keep at 72°C for 2 minutes, then run a Final Hold at 4°C. | |||
'''Section 2: Research and Development''' <br> | |||
: 1. | |||
:: a. The function of template DNA is to act as the basic template for which thousands of complementary strands can be made. | |||
:: b. Primers bind to each end of the template DNA and make copies of the original DNA. | |||
:: c. The function of Taq Polymerase is to actually synthesize these strands of complementary DNA. | |||
:: d. Deoxyribnucleotides are the basic building blocks which form the basic components of the strands. | |||
2. | : 2. | ||
:: a. In the initial step, the DNA is heated to bring it close to denaturation. | |||
:: b. Then, the DNA is denatured and left single-stranded to expose the base pairs. | |||
:: c. During annealing, the two primers bind to the appropriate end of a strand. | |||
:: d. Taq polymerase extends the primer. | |||
:: e. Final elongation occurs to any previously unextended strands. | |||
:: f. This is a short-term storage hold of the reaction. | |||
: 3. Adenine (A) and Thymine (T) pair together. Cytosine (C) and Guanine (G) pair together. | |||
: 4. Base-pairing occurs during the extending step and final step, when elongation occurs and Taq polymerase synthesizes the strands. |
Latest revision as of 09:45, 29 October 2014
BME 100 Fall 2014 | Home People Lab Write-Up 1 | Lab Write-Up 2 | Lab Write-Up 3 Lab Write-Up 4 | Lab Write-Up 5 | Lab Write-Up 6 Course Logistics For Instructors Photos Wiki Editing Help | |||||||||||||||||||||||||||||||||
Lab 4: PCR DNA Amplification
PCR Reaction Sample List:
DNA Sample Set-Up Procedure
Run the heating and cooling program on the thermal cycler.
Section 2: Research and Development
|