PCR Protocol

In a traditional PCR protocol, reaction components are assembled as described below. The final volume should be 50 µL.

  • Thaw all reagents on ice.
  • Assemble reaction mix into 50 µL volume in a thin walled 0.2 mL PCR tubes.
  • Add reagents in following order: water, buffer, dNTPs, Mg CL2, template primers, Taq polymerase.
  • Gently mix by tapping tube. Briefly centrifuge to settle tube contents.
  • Prepare negative control reaction without template DNA.
  • Prepare positive control reaction with template of known size and appropriate primers.
  • Component
    Final Concentration/amount
    water
    to 50 µL
    buffer
    1 x
    Taq polymerase
    0.05 units/µL
    dNTP mix
    200 µM
    MgCl2
    0.1-0.5 mM
    Forward primer
    0.1-0.5 µM
    Reverse primer
    0.1-0.5 µM
    template
    200 pg/µL
    DMSO (optional)
    1 to 10%  w/v

PCR Steps

  • Program your thermocycler for your PCR reaction using the following guidelines:
    PCR protocol
    Step Temp Time # of cycles
    Initial Denaturation
    94°C
    5 min
     
    Denaturation
    94°C
    30 sec
    30-35
    Primer Annealing
    Tm-5°C
    45 sec
    Extension
    72°C
    1 min per kb
    Final Extension
    72°C
    5 min
     
  • Analyze the results of your PCR reaction via gel electrophoresis.

PCR Design Tools

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