1x recipe for a 50uL PCR reaction

  • 10uL HF buffer
    • HF buffer should be 5x or adjust volume so final conc. of HF is 1x.
  • 1uL dNTP's
    • This can vary depending on conc. the final conc. should be 0.2mM.
  • 3uL of forward primer
  • 3uL fo reverse primer
    • The primer volume can differ quite a lot depending on which conc. you have them in. The final conc. of the each primer should be in the range of 1uM to 0.1uM but this can also be hard to generalize.
  • 0.5uL of polymerase
    • This again depend on the unit per microliter conc. of the polymerase. Use approx. 2.5U per reaction.
  • 1uL template
    • The conc. of template can vary and this can also be one of the variables to turn on if the PCR goes wrong.
  • 31.5uL MQ-water
    • If using more or less of the previous reactant then just adjust the MQ amount so that the final volume will be 50uL

To lessen the work and use of pipette tips you can make a PCR-mastermix with the following components:

PCR mastermix

  • 10uL HF buffer
  • 1uL dNTP's
  • 31.5uL MQ
  • 0.5uL polymerase
    • Add this as last step as this is best for the polymerase activity.