PCR Laboratory Notes

PCR (Polymerase Chain Reaction) Overview
  • Objective: Enable students to perform and understand the PCR process. Specific goals include:
    • Describe the cycling conditions and processes at each temperature.
    • Identify PCR components and their functions.
    • Accurately micropipette small volumes for PCR setup.
    • Locate PCR primers within a template DNA.
    • Predict size and DNA in the PCR product.
Basics of PCR
  • Steps in PCR:
    1. Denaturing: Heating the DNA to separate strands.
    2. Annealing: Cooling down to allow primers to bind to each strand.
    3. Extending: DNA polymerase builds a new strand from the primers.
  • Process repeats to double the amount of DNA with each cycle.
PCR Procedure Overview

  1. Preparation:

    • Use a 1.5 mL tube for the PCR master mix, kept on ice.
    • The master mix accommodates 3 reactions (1 for each student, 1 negative control).

  2. Reagent Setup: Use the following volumes for the master mix:

    ReagentVolume (μL)
    Sterile Distilled H2O165
    10X PCR Buffer20
    10 mM dNTP4
    DNA Primer 14
    DNA Primer 24
    Taq Polymerase (added by TA)1

  3. Adding Reagents:

    • Micropipette 165 μL dH2O into the tube.
    • Add 20 μL of 10X PCR buffer.
    • Change the pipet tip between solutions to avoid contamination.
    • Add 4 μL of the 10 mM dNTP mixture and then the primers (4 μL each).
    • Notify the instructor to add Taq polymerase while keeping the mix on ice.

  4. Mixing:

    • Mix the solution gently by pipetting to avoid bubble formation.
    • Transfer 47 μL of the master mix into three labeled PCR tubes (yours, partner's, and negative control).
    • Add 3 μL of cheek cell DNA to your and your partner's tubes and 3 μL water to the negative control tube.

  5. Final Mixing and PCR Cycling:

    • Mix each tube, then place them into a thermal cycler programmed for 35 cycles:
      • Denaturing: 94°C for 45 seconds.
      • Annealing: 58°C for 30 seconds.
      • Extending: 72°C for 1 minute.
    • Upon completion, samples will be held at 4°C and stored at -20°C.
Cleanup and Safety
  • Keep reagents on ice throughout the procedure.
  • Dispose of used tips and tubes in designated waste.
  • Wipe down benches and wash hands after the experiment.
  • Duration: PCR takes about 2 hours.
  • DNA will remain frozen until further experimentation is conducted.