Lab 4: PCR DNA Amplification
Purpose/Goal: To successfully amplify a section of DNA containing a disease marker in order to accurately diagnose patients.
- a. Lab coat and disposable gloves
- b. PCR reaction mix, 8 tubes, 50 uL each: Mix containts Taq DNA polymerase, MgCl2, and dNPT's
- c. DNA/primer mix, 8 tubes, 50 uL each: Each mix contains a different template DNA. All tubes have the same forward primer and reverse primer.
- d. A strip of empty PCR tubes
- e. Disposable pipette tips: only use each one once. Never re-use disposable pipette tips or samples will be cross-contaminated.
- f. Cup for discarded tips
- g. Micropipettor
- h. OpenPCR machine: shared by two groups
PCR Reaction Sample List:
| Tube Label
|| PCR Reaction Sample
|| Patient ID
| G27 P
|| Positive control
| G27 N
|| Negative control
| G27 1-1
|| Patient 1, replicate 1
| G27 1-2
|| Patient 1, replicate 2
| G27 1-3
|| Patient 1, replicate 3
| G27 2-1
|| Patient 2, replicate 1
| G27 2-2
|| Patient 2, replicate 2
| G27 2-3
|| Patient 2, replicate 3
DNA Sample Set-Up Procedure
- 1. Without opening the tubes, obtain patient samples along with the positive and negative controls. Each set has its own patient ID number, and there should be 3 copies of each patient sample.
- 2. Create the report by replacing the '#' with your group's number (27) and then record the Patient IDs in your table/list. Remember to not actually write anything on the tubes.
Basic methodology behind PCR practice. Separate strands chemically, then synthesize complementary DNA strands from the original templates with polymerases. Repeat ad nauseum.
Run the heating and cooling program on the thermal cycler.
- 1. Heating Lid at 100°C
- 2. Keep at 95°C for about 2 minutes
- 3. Run 35 cycles, then denature at 95°C for 30 seconds.
- 4. Anneal at 57°C for 30 seconds, then extend at 72°C for 30 seconds.
- 5. Finally, keep at 72°C for 2 minutes, then run a Final Hold at 4°C.
Section 2: Research and Development
- a. The function of template DNA is to act as the basic template for which thousands of complementary strands can be made.
- b. Primers bind to each end of the template DNA and make copies of the original DNA.
- c. The function of Taq Polymerase is to actually synthesize these strands of complementary DNA.
- d. Deoxyribnucleotides are the basic building blocks which form the basic components of the strands.
- a. In the initial step, the DNA is heated to bring it close to denaturation.
- b. Then, the DNA is denatured and left single-stranded to expose the base pairs.
- c. During annealing, the two primers bind to the appropriate end of a strand.
- d. Taq polymerase extends the primer.
- e. Final elongation occurs to any previously unextended strands.
- f. This is a short-term storage hold of the reaction.
- 3. Adenine (A) and Thymine (T) pair together. Cytosine (C) and Guanine (G) pair together.
- 4. Base-pairing occurs during the extending step and final step, when elongation occurs and Taq polymerase synthesizes the strands.