LAB 4 WRITE-UP
- Lab Coat
- Disposable Gloves
- Cup to discard tips
- Disposable pipette tips
- Strip of empty PCR tubes
- 8 tubes of PCR reaction mix, each containing 50 μL.
- 8 tubes of DNA/ Primer mix, each containing 50 μL.
- OpenPCR machine
PCR Reaction Sample List
||PCR Reaction Sample
||Patient 1, replicate 1
||Patient 1, replicate 2
||Patient 1, replicate 3
||Patient 2, replicate 1
||Patient 2, replicate 2
||Patient 2, replicate 3
DNA Sample Set-up Procedure
- Cut the PCR tubes in half so that it can fit into the machine
- Label the sides of the tubes with the tube labels with a black marker
- Place tubes onto rack
- Begin with the empty tube labeled as the positive control. Using the correct micropipette technique, transfer 50 microliters of the PCR reaction mix into the empty tube. Be sure not to cross-contaminate the tips by discarding every time a substance is transferred.
- Using a fresh pipette tip, transfer 50 microliters of the positive control DNA primer mix into the same tube. The total volume of the tube should be 100 microliters.
- Repeat steps 5 and 6 for the negative control, patient 1 replicate 1,2 and 3 and patient 2 replicate 1,2 and 3. Use the correct DNA/Primer mix for the corresponding tubes. Upon completion, all labeled tubes should contain 100 microliters of the DNA primer mix and the PCR mix
- Close the lids tightly on the PCR reaction tubes
- Place the tubes in the PCR machine. All 16 slots should be filled and run the trial.
The OpenPCR machine runs a heating and cooling program on the thermal cycler. The heated lid is heated to 100 ℃. The machine then goes through the initial set up at 95 ℃ for 2 minutes. After completing the initial step, it completes 35 cycles. During one cycle, the samples denature at 95℃ for 30 seconds, Anneal at 57℃ for 30 seconds and finally denature at 72 ℃ for 30 seconds. After this is completed, the final step runs at 72℃ for 2 minutes and the final hold is held for 4℃.
Research and Development
PCR - The Underlying Technology
Amplification of a DNA sequence usually involves 20-30 cycles of PCR and each cycle requires 3 temperature changes, which are completed automatically in a thermocycler. The initial step of the PCR machine lasts for 3 minutes at 95℃ and it ensures that the template DNA is denatured into 2 separate template DNA strands. After the initial step, usually 20-30 cycles of denaturing, annealing and extension occur. The denature stage occurs at 95℃ and it results in the template DNA being split into 2 single stranded DNA molecules. The next step, annealing, requires lowering the temperature to 57℃ to allow for the primers to attach to the appropriate nucleotides on both the DNA strands. This step usually requires 30 seconds to complete. After annealing, a 30 second step, Extension occurs. The temperature increases to 72℃. During this step, Taqpolymerase binds to the primer and adds dNTPs to the single stranded DNA molecule to create two double stranded DNA molecules. The Final step occurs at 72℃ for 3 minutes. This step continues to extend the DNA molecules to make sure they are fully formed. The Final hold occurs at 4℃ to ensure that bonding occurs within the DNA strands.
Base Pair Bonding
Adenine and Thymine bond together and Guanine and Cytosine Bond together.