BME100 f2014:Group11 L4
- Lab Coat and disposable gloves
- OCR reaction mix, 8 tubes, 50μL each: Mix contains Taq DNA polymerase, MgCl2, and dNTP's
- DNA/primer mix, 8 tubes, 50μL 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.
- Cups for discarded tips
- OpenPCR machine: shared by two groups
|Tube Label||PCR Reaction Sample||Patient ID|
|G#11 P||Positive Control||None|
|G#11 N||Negative Control||None|
|G#11 1-1||Patient 1, replicate 1||81166|
|G#11 1-2||Patient1, replicate 2||81166|
|G#11 1-3||Patient1, replicate 3||81166|
|G#11 2-1||Patient2, replicate 1||54283|
|G#11 2-2||Patient2, replicate 2||54283|
|G#11 2-3||Patient2, replicate 3||54283|
Open PCR Program HEATED LID: 100°C
INITIAL STEP: 95°C for 2 minutes
NUMBER OF CYCLES: at least 20 but ideally 35. Denature at 95°C for 30 seconds, Anneal at 57°C for 30 seconds.
FINAL STEP: 72°C for 2 minutes
FINAL HOLD: 4°C
Sampple DNA Set- up
1) Collect all required materials listed above.
2) Cut the empty PCR tube strips in half so that there are two strips of four empty tubes.
3) Label the sides of the tubes with your own tube labels and place tubes on rack.
4) Starting with your tube labeled the positive use the micro-pipette to transfer 50 μL PCR reaction mix into this tube and then discard the disposable tip into the plastic cup.
5) With a new pipette tip transfer the positive control mix into the same tube. The final mixture should be 100 μL. Discard the tip.
6) Repeat these steps with the remaining seven tubes-- the negative control, patient one DNA replicates 1,2 and 3, patient two DNA replicates 1,2 and 3, in the corresponding tubes. When you're done, the tubes should be a combination of the PCR reaction mix and the DNA.
7) Make sure the lids are closed and place tubes in the PCR machine and with the sixteen slots full run the machine.
Research and Development
PCR is method used to copy DNA that implements cycles of heating and cooling. In this process, template DNA is used to provide the pattern for the sequence of nucleotides of a RNA transcript. This template is heated to 95 degrees Celsius for three minutes so that the DNA denatures into two strands.It should take about 30 seconds for the DNA to denature.The temperature is then dropped to 57 degrees Celsius for another 30 seconds. At this point primers bind to specific sections of the DNA strands and it is at these primers that the copying starts. The temperature raises to 72 degrees Celsius for 30 more seconds to allow the enzyme TAQ polymerase to bind to the primers and add the missing, complementary nucleotides to the strands of DNA. This cycle is repeated for at least 35 cycles, or until there are enough copies of the DNA segment. At the end of the entire process is where Deoxyribonucleotides come in. dNTP's are the building blocks of DNA and are necessary for DNA to be synthesized as they create the final bonds. For this step the temperature is kept at 72 degrees Celsius for 3 minutes. Finally, the temperature drops to 4 degrees Celsius until the tubes are removed from the PCR machines and stored in a refrigerator.