STEP 2: Design primer
Generally, primers used are 18-23 mer in length. Use Primer3 free online software or other assistant tools like Primer Premier to design proper primers. Choose the best primer pairs  from those options according to the parameters, such as size, TM, GC%, etc.
STEP 3: Determine annealing temperature:
Melting temperature (Tm) of primers can be calculated by the following formula:
Tm = [[(Tm_Forward +Tm_Reverse)×1/2]-10]°C.
Tm°C is a good annealing temperature to start with. However, optimal annealing temperatures can only be determined experimentally for a certain primer/template combination. Temperature gradient PCR is often a way to finalize an optimal annealing temperature.
STEP 4: For a 50 μl reaction, add:

10x PCR buffer	5 μl
DNA template	X μl
Forward Primer	1 μl
Reverse Primer	1 μl
dNTPs Mix	1 μl
Taq Polymerase	0.25μl
Sterile ddH2O	To 50μl

 Notes:
1.Primers diluted to working concentration (10µM working stocks are sufficient for most assays).
2.Final Concentration of primers should be 0.1-0.5 µM, final Concentration of dNTPs Mix should be 200µM, final concentration of DNA template should be 200 pg/µL
3. Mix gently by vortex and briefly centrifuge to collect all components to the bottom of the tube.
STEP 5: A typical PCR program                    

CYCLE STEP	TIME	TEMP	CYCLES
Initial denaturation	30 sec	95 °C	1
Denaturation Annealing Extension	15-30 sec  15 sec depends on length, 1 min/ kb	95 °C depends on Tm 72℃	30
Final extension	5 min	72 °C	1
Keep sample	∞	4 °C	-

Notes:
The amplification parameters will vary depending on the primers and the thermal cycler used. It may be necessary to optimize the system for individual primers, template, and thermal cycler.