Polymerase Chain Reaction

Polymerase Chain Reaction (PCR)

The system being utilised will influence the optimal operating conditions for the concentration of Taq DNA Polymerase, template DNA, primers and MgCl2. It may be necessary to determine the optimal concentration of MgCl2 and cycling parameters.Taq DNA Polymerase and MgCl2 can be titrated to determine the optimum efficiency. Below is a general PCR protocol for a 50 µl reaction with some recommendations.

Reagents

  • 5 µl 10X Standard Taq Reaction buffer
  • 1 µl 10 mM dNTPs
  • 1 µl 10 mM Forward Primer
  • 1 µl 10 mM Reverse Primer
  • Template DNA
  • 25 µl Taq DNA Polymerase
  • Make up to 50 µl Nuclease-free water

Procedure

  1. Preheat the thermocycler to 95oC.
  2. Gently mix the reaction components listed above. Spin quickly to collect the liquid to the bottom of the tube if necessary.
  3. Overlay the sample with mineral oil if using a PCR machine without a heated lid.
  4. Transfer PCR tubes from ice to the preheated PCR machine (95oC) for the initial denaturation step.
  5. Begin thermocycling:
    • Initial denaturation: 95 °C, 30 seconds
    • 30 cycles of: 95 °C, 15-30 seconds; 45 °C – 68 °C, 15 – 60 seconds; 68 °C, 1 minute/kb
    • Final extension: 68 °C, 5 minutes
    • Hold: 4 – 10 °C
  6. Validate the final reaction mixture by running a fraction on an agarose gel with an appropriate molecular marker, directly sequence the amplified product or carry out restriction enzyme digests.

Recommendations

  • MgCl2: For most Taq DNA Polymerase a MgCl2concentration of 1.5 – 2.0 mM is optimal. The final concentration in standard 1X Standard Taq Reaction Buffer is 1.5 mM. If another concentration is optimal, a reaction buffer that does not contain MgClcan be used and the MgClcan be added separately.
  • Additives: DMSO and formamide can be added to improve difficult targets such as GC-rich sequences.
  • Template DNA: Use high quality, purified DNA. For a 50 µl reaction, recommended amounts are:
    • Genomic DNA 1 ng – 1 µg
    • Plasmid or viral DNA 1 pg – 1 ng
  • Taq DNA Polymerase: 1.25 units/50 µl reaction is recommended, however this can vary from 0.25 – 2.5 units/50 µl reaction.
  • Denaturation: A longer initial denaturation step (2 – 4 minutes at 95 °C) can be employed for difficult templates such as GC-rich sequences. For colony PCR 5 minutes at 95 °C is recommended. During themocycling 15 – 30 seconds at 95 °C is recommended.
  • Annealing: The temperature is dependent on the Tof the primer pair (usually 45 °C – 68 °C). Optimise by carrying out temperature gradient PCR starting 5 °C below the TM.
  • Extension: The recommended temperature is 68 °C with the time generally 1 minute per kb. A final extension of 5 minutes at 68 °C is recommended.
  • Cycle number: Generally, 25 – 35 cycles yields sufficient product. Up to 45 cycles can be employed to detect low copy number targets.