BME100 s2015:Group4 9amL4

BME 100 Spring 2015	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
OUR TEAM Name: Measho Habtemichael Role(s) Name:Aaron Frisby Role(s) File:Cody manning.jpg Name: Cody Manning Role(s) Name: Alexandra Torres Role(s) Name: Amanda Nguyen Role(s) LAB 4 WRITE-UP Protocol Materials Lab coat and disposable gloves PCR reaction mix, 8 tubes, 50 μL each: Mix contains Taq DNA polymerase, MgCl2, and dNTP’s DNA/ primer mix, 8 tubes, 50 μL each: Each mix contains a different template DNA. All tubes have the same forward primer and reverse primer A strip of empty PCR tubes Disposable pipette tips: only use each only once. Never re-­‐use disposable pipette tips or samples will be cross-­‐contaminated Cup for discarded tips Micropipettor OpenPCR machine: shared by two groups PCR Reaction Sample List Tube Label PCR Reaction Sample Patient ID G4 + Positive control none G4 - Negative control none G4 1-1 Patient 1, replicate 1 19902 G4 1-2 Patient 1, replicate 2 19902 G4 1-3 Patient 1, replicate 3 19902 G4 2-1 Patient 2, replicate 1 33338 G4 2-2 Patient 2, replicate 2 33338 G5 2-3 Patient 2, replicate 3 33338 DNA Sample Set-up Procedure Retrieve all materials In the eight patient tubes, ensure there is the PCR reaction mix Label each of the tubs according to the PCR Reaction Sample List In the negative control tube, add 50 microliters of negative control In the positive control tube, add 50 microliters of positive control Then in the three tubes for patient 1, add 50 microliters of patient 1's DNA mix In the three tubes for patient 2, add 50 microliters of of patient 2's DNA mix Close all of the tubes Place in OpenPCR machine OpenPCR program HEATED LID: 100°C INITIAL STEP: 95°C for 2 minutes NUMBER OF CYCLES: 35 Denature at 95°C for 30 seconds, Anneal at 57°C for 30 seconds, and Extend at 72°C for 30 seconds FINAL STEP: 72°C for 2 minutes FINAL HOLD: 4°C Research and Development PCR - The Underlying Technology There are many components to a PCR reaction. A full reaction requires a DNA template, a DNA polymerase, primers, and nucleotides. The DNA template is a sample of DNA that contains the sequence that needs to be analyzed. This template is then manipulated in order to separate the strands of DNA from each other. DNA polymerase is an enzyme that creates complementary strands of DNA to the target sequence of DNA. The polymerase used in this reaction is Taq DNA polymerase. Primers are small pieces of single strands of DNA. These small pieces of DNA form together to create the complimentary strand of DNA. The polymerase then begins to create the new strands starting at the end of the primer. Nucleotides are the basic building blocks of DNA. They are the bases A, T, G, and C. These building blocks are used by the polymerase and primer in order to create the complementary strand of DNA. Source: http://www.ncbi.nlm.nih.gov/genome/probe/doc/TechPCR.shtml During the INITIAL STEP, the DNA is heated to 95*C in order to begin the denaturing process. During the DENATURE step, DNA is heated even further in order to separate the double strands of DNA. The heat causes the hydrogen bonds within DNA to break, releasing the two strands. During the ANNEAL step, the temperature is lowered in order to allow the primers to adhere to the now single strands of DNA. During the EXTEND step, the temperature is increased slightly. At this temperature, the polymerase then begins working with the primers in order to begin to create complimentary strands of DNA. The FINAL STEP is to ensure that all complementary nucleotides have been attached to the complimentary strand. The FINAL HOLD is used for an indefinite period of time in order to maintain the complimentary strands of DNA until it is time for the strands to be used. Adenine (A) bonds to Thymine (T) Thymine (T) bonds to Adenine (A) Cytosine (C) bonds to Guanine (G) Guanine (G) bonds to Cytosine (C)