BME100 s2014:T Group14 L4

BME 100 Spring 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
OUR TEAM Name: Caitlin R. Byrne Name: Megan McGuire Name: Wade Savage Name: Theo Hall LAB 1 WRITE-UP Initial Machine Testing The Original Design OpenPCR Machine Source: http://makezine.com/2011/07/06/dna-is-now-diy-openpcr-ships-worldwide/ A PCR machine is basically a photocopier for DNA. It splits apart the double helix by heating in order to make copies of it. For a polymerase chain reaction to take place a particular DNA sequence should be identified and then amplified by creating up to millions of copies of the specific strands. A PCR machine can go through many cycles of this process and can turn a strand of DNA into thousands of copies of the exact same single strands of DNA. The OpenPCR uses a display on the actual device and a USB port to connect to the computer in order to change the parameters of the PCR reaction we want to take place. The OpenPCR machine is basically a "Do-It-Yourself" kit for anyone to buy, make, and then use. Since it's you have to put together, this equipment is open to some errors if not put together right. This is why we tested the PCR machines before conducting our experiment. Experimenting With the Connections When we unplugged (part 3) from (part 6), the machine's display turned off; this is the power cable for the display. When we unplugged the white wire that connects (part 6) to (part 2), the machine's display showed an incorrect temperature. We assume that this is the wire that connects the display and internal computer to the integral part of the device that measures the temperature of the PCR machine. Test Run We preformed our test run of our OpenPCR machine on Thursday March 20, 2014. Set up was simple and went smoothly, we placed the empty test tubes into the machine and ran the machine. After setting up everything correctly, our machine ended up failing the test. The computer program projected that our the time for our reaction would extend way beyond the two hour time period and didn't even finish 1 cycle. This resulted in our machine's failure and was noted to not be used during the experiment. Protocols Thermal Cycler Program Original image of our group's inputs into the OpenPCR software. Thermal Cycler Program Make sure the OpenPCR machine is connected to the computer through the USB cord. Start Open PCR Software Select "Add a New Experiment" Click "More Options" to begin inputing the specifications of the cycles the PCR machine will go through. Enter Program Parameters: 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 Extend at 72°C for 30 seconds FINAL STEP: 72°C for 2 minutes FINAL HOLD: 4°C DNA Sample Set-up This is the test tube set up we inserted into the OpenPCR system. We had two different subjects of DNA, one female and one male, and we labeled everything as follows on our 8 50 microliter test tubes. Patient 1 Patient 2 Controls Sample 1 1a 2a dDNA (Positive Control) Sample 2 1b 2b ndDNA Sample 3 1c 2c dDNA -- the disease DNA ndDNA -- the non-disease DNA female subject -- 1a, 1b, 1c male subject -- 2a, 2b, 2c DNA Sample Set-up Procedure 1. Collect materials needed, which include: the PCR reaction mix in 8 test tubes with 50μL in each, 8 empty attached PCR tubes, 8 tubes of the Template DNA and the primer mix combined with 50μL in each tube, a box of disposable pipette tips, and a 200μL micropipettor. 2. Then cut the tubes in sets of 4. You need to do this in order for the tubes to fit in the OpenPCR machine. 3. Then the tubes will be labeled with the predetermined labels we named above with a permanent marker. 4. The tubes are placed in a test tube rack after being labeled. 5. Attach a tip to the micropipettor, and transfer 50μL of the PCR reaction mix into the positive control. Then, dispose the tip into the proper waste disposal. 6. Transfer the positive disease containing DNA and primer mix into the same tube and use a new tip and dispose it properly. 7. Repeat steps 5 and 6 for the negative control, patient 1, and patient 2, remembering to dispose the tip after each substance in each PCR tube. In total 100μL will be each test tube. 8. Place the tubes into the PCR machine, set up the parameters needed for the test run. 9. Get help from the TA in order to start the machine. PCR Reaction Mix What is in the PCR reaction mix? Taq DNA polymerase, MgCl2, and dNTP's. DNA/ primer mix What is in the DNA/ primer mix? template DNA, and the same forward primer and reverse primer. Research and Development PCR - The Underlying Technology (Add a write-up, essay-style, organized into paragraphs with descriptive headers, based on the Q&A's from Section three of your worksheet) (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 PCR video/ tutorial might be useful. Be sure to credit the sources if you borrow images.)