Q5 PCR

Materials

  • 50µL Reaction
    • 0.5µL Q5 DNA polymerase
    • 2.5µL 10µM forward primer
    • 2.5µL 10µM reverse primer
    • 10µL 5x Q5 reaction buffer
    • 0.5µL template DNA
    • 1µL dNTP mix (10mM each)
    • 33µL ddH2O

    Setup

    1. Mix everything together

    Master Mix

    reagentconcentrationpipet this much
    forward primer (µM)102.5
    reverse primer (µM)102.5
    5x Q5 rxn buffer10
    template DNAdoesn't matter!0.5
    dNTP mix (mM each)101
    Q5 polymerase0.5
    water"=B10*B11-sum(C2:C7)" -->33
    "=(B10*B11)-sum(C2:C7)" -->33
    how many reactions are you doing?2
    how many µL do you want each reaction to be?25

    Thermal cycler program

    1. 98C for 5min
    1. 98C for 10s
    1. 55C for 30s
    1. 72C for 20s/kb

    minimum time 30s

    1. Repeat steps 2-4 (3-5?) 29 times
    1. 72C for 10min
    1. 10C hold
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