LAB 4 WRITE-UP
- 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
- OpenPCR machine: shared by two groups
PCR Reaction Sample List
| Tube Label
|| PCR Reaction Sample
|| Patient ID
| G7 +
|| Positive control
| G7 -
|| Negative control
| G7 1-1
|| Patient 1, replicate 1
| G7 1-2
|| Patient 1, replicate 2
| G7 1-3
|| Patient 1, replicate 3
| G7 2-1
|| Patient 2, replicate 1
| G7 2-2
|| Patient 2, replicate 2
| G7 2-3
|| Patient 2, replicate 3
DNA Sample Set-up Procedure
- Make sure that your group has all of the Materials listed in the protocol you wrote in Lab A.
- Cut the strip of empty PCR tubes in half so that you have two strips of four linked tubes. This is
necessary to fit all of your tubes into the OpenPCR machine.
- Use a black marker to label the sides of the empty tubes with the Tube!Labels your group created. Do not label the lids of the tubes!
- Place the tubes in a rack.
- Start with the empty tube you labeled as the positive control. Using proper pipetting technique, transfer 50 μL of PCR reaction mix into this empty tube. Discard the disposable tip into the collection cup. Do not reBuse tips and crossBcontaminate your samples!
- Using a fresh pipette tip, transfer the positive control DNA/ primer mix into the same tube. The total volume in your positive control PCR reaction tube is now 100 μL.
- Repeat steps 5 and 6 for the negative control, patient 1 replicates 1, 2, and 3, and patient 2 replicates 1, 2, and 3. Use the appropriate DNA/ primer mix for the corresponding tubes. When you are done, all of your labeled tubes should contain DNA/ primer mix plus PCR mix, resulting in a 100!μL!complete! PCR!reaction in each tube.
- Close the lids tightly on your PCR reaction tubes.
- Take the tubes over to your assigned PCR machine. Place the tubes into the slots in the heating block. Do not start the machine until all 16 slots are filled (multiple groups will need to run reactions simultaneously). If you are unsure whether it is okay to start a run, ask a TA for help.
- 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