Colony PCR

Performed with Thermo Scientific Taq DNA Polymerase (recombinant), 5U/μl

	Volume (μl)
10×Taq Buffer	5
dNTP Mix, 2 mM each	5 (0.2 mM of each)
Forward primer	0.1-1.0 μM
Reverse primer	0.1-1.0 μM
25 mM MgCl2*	1-4 mM
Template DNA	Picked from the colony after transformation
Taq DNA Polymerase	0.25 U
ddH2O	To 10
Total	10

*Volumes of 25 mM MgCl2, required for specific final MgCl2 concentration:

Final concentration of MgCl2(mM)	1	1.5	2	2.5	3	4
Volume of 25 mM MgCl2 to be added for 10 μl reaction(μl)	0.4	0.6	0.8	1.0	1.2	1.6

3. Gently vortex the samples and spin down.
4. Perform PCR using recommended thermal cycling conditions:

Step	Temperature, °C	Time	Number of cycles
Initial denaturation	95	10 min	1
Denaturation	95	30s	25~40
Annealing	Tm-5	30s	25~40
Extension	72	1 min/kb	25~40
Final Extension	72	5-15 min	1