Korean Journal of Breeding Science

Conditions of multiplex-PCR for identification of Korean red winter wheat cultivars

Multiplex-PCR	PCR1	PCR2	PCR3
Allele	Ppo-A1 Rht-B1b Vrn-D1	TaCwi-A1b Glu-A3ac Lr34	Glu-A1c Glu-B1b KWSM003 TaSE96
PCR reaction components
Taq DNA polymerase (U)	2	4	4
Each dNTP (mmol/l)	3	4	3
Primer (μmol/l)	1/1/2z	0.5/1.5/1.5y	2/1/1/1x
Concentration of DNA (ng)	120	100	80
PCR amplification condition
C	94℃, 5 m	94℃, 5 m	94℃, 5 m
Denaturation	94℃, 1 m	94℃, 1 m	94℃, 1 m
Annealing	62℃, 1 m	62℃, 1 m	64℃, 1 m
Extension	72℃, 1 m	72℃, 1 m	72℃, 1 m
No. of cycles	35	33	33
Final extension	72℃, 10 m	72℃, 10 m	72℃, 10 m
Gel electrophoresis
Agarose (%)	1.5	1.5	1.5
Voltage (V)	135	135	135
Time (h)	1	1	1

^zConcentrations of primers for Ppo-A1, Rht-B1b and Vrn-D1a, respectively.

^yConcentrations of primers for TaCwi-A1b, Glu-A3ac and Lr34, respectively.

^xConcentrations of primers for Glu-A1c, Glu-B1b, KWSM003 and TaSE96, respectively.