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. Pfx elongates at a rate of 1kb (1000bp) per 30s. Look up the length of

1. the 

   gene of interest and calculate time of elongation.

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

   temperature will be that number minus 2

1. .

Assembling Reaction

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

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

3. Thaw Pfx supermix on ice.

4. Add the following (in order):
    

   
   

   VOLUME	REAGENT
   22.5uL (for 35 cycles)	Pfx Supermix
   2uL	5uM Forward Primer
   2uL	5uM Reverse Primer
   1uL	Template DNA(~150ng)

Programming The Thermocycler

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 30s per kb

Final Elongation:

 72C for 10min
Store:

 4C