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Things to keep in mind!

  • Annealing temperature of primers (Tm) should be around 60 C
  • Check the secondary structure of the primers before you order them!
    • no individual secondary structures i.e. hairpins
    • no heterostructure with the forward and reverse primers together
    • free energy of primers should be greater than -4 kCal 
    • GC content should be around 50% (40-60% is okay) 

  1. Dilute your DNA to the following concentrations:

    DNAConcentrations
    Template0.1 - 1 ng/ul
    Forward Primer10 uM
    Reverse Primer10 uM
  2. Set up a small box (e.g. empty pipette tip box) with ice and water. Your DNA and polymerase mix will go into this box before going into the the thermocycler in order to limit endonucelase activity. 

  3. Add the following DNA to a labeled 0.6ml PCR tube 

    DNAVolume
    Template1 uL
    Forward Primer500 nM
    Reverse Primer500 nM

  4. Program the thermocycler as follows

    TemperatureTime
    9830s
    PAUSE
    985s
    Tm15
    72(15s)x(#kb)
    725m
    4forever

  5. Wait for thermocycler to heat up 

  6. Add 22.5uL of polymerase mix (Phusion Master Mix) to your DNA. Mix well and spin down. Transfer tubes to ice as soon as possible. 
  7. Once the thermocycler has heated up to the right temperature (it should be paused at 98C), add tubes to thermocycler and resume PCR program. 


 

Calculating Reaction Conditions

  1. Use idtdna.com or VectorNTI to calculate melting temperatures of primers

    without common overhangs (base pairs 30 to end when read 5' to 3').

     

    PRIMER

    Tm

    FW

     

    RV

     

  2. Phusion elongates at a rate of 1kb (1000bp) per 15s. Look up the length of the 

    gene of interest and calculate time of elongation.

  3. You should get your Tm from NEB.

    If you get the melting temperature of your primer from Genious, the annealing

    temperature will be that number minus 2.

Assembling Reaction

  1. Get 0.6mL PCR tubes (not the strip tubes).

  2. Get primers for gene of interest. Resuspend if necessary.

  3. Thaw Phusion supermix on ice.

  4. Add the following (in order):
     


    VOLUME

    REAGENT

    22.5uL (for 35 cycles)

    Phusion Supermix

    2uL

    5uM Forward Primer

    2uL

    5uM Reverse Primer

    1uL

    Template DNA(~150ng)

     

Programming The Thermocycler

(In the 3rd floor thermocycler, the PCR program is named "PHUSION")

Initial Denaturation: 98C for 5min

LOOP: 30-35 cycles

CYCLE: 
Denaturation: 98C for 10s
Annealing: calculated temperature (typically 55-65C) for 30s
Elongation: 72C for 15s per kb

Final Elongation: 72C for 10min
Store: 4C