TABLE 2.

PCR conditions and thermocycling profiles used for canine Slc11a1 sequencing and polymorphism analysis

Region	Primers	MgCl2concn (mM)	Primer concn (μM)	Conditions for:						No. of cycles	Enzyme(s)a
				Denaturation		Annealing		Extension
				°C	s	°C	s	°C	s
Promoter 1	NRPROM-F-NRI2E2-R	1.50	0.2	94	30	55	30	72	60	35	A
	NRPROM3-F-NRPROM2-R	1.50	0.2	94	30	54	30	72	60	35	A
Promoter 2	NRPROMD-F-NRPROM3-R	1.50	0.2	94	30	53	30	72	60	35	A
	NRPROMD-F-NRPROM2-R	1.50	0.2	94	30	53	30	72	60	35	A
E2-E7	NRE2-F-NRE7-R	2.25	0.3	92	10	58	30	68	240	10	B
				92	10	58	30	68	240b	25
E2-E9	NRE2-F-NRE9-R	1.50	0.2	94	30	53	30	72	60	35	A
E7-E15	NRE7-F-NRE3′UTR-R	1.50	0.2	94	30	55	30	72	60	35	A
E6-I6	NRE6I6-F-NRI6-R	2.00	0.3	94	30	63c	30	72	30	20	D
				94	30	53	30	72	30	10
E7-E10	NRE7-F-NRE10-R	1.50	0.3	94	15	55	30	68	480	35	C
E9-E11	NRE9-F-NRE11-R2	1.50	0.3	94	15	55	30	68	480	35	C
E10-E15	NRE10-F-NR3′UTR-R	1.50	0.3	94	15	55	30	68	480	35	C
Microsatellite	NRMICRI1-F-NRMICR1-R	1.50	0.2	94	30	55	30	72	30	25	A
Intron 10	NRE10-F-NRE11-R2	1.50	0.2	95	30	55	30	72	45	35	E

^aEnzymes and commercial kits: A, Taq polymerase (Invitrogen); B, Expand long-template PCR system (system 3; Taq and Pwo polymerases) (Roche); C, Platinum Pfx DNA polymerase (with enhancer solution ×1) (Invitrogen); D, AmpliTaq Gold (Applied Biosystems); and E, GC-Rich PCR amplification system (0.5 M GC rich resolution solution and Taq and Tgo polymerases).

^bPlus 20 s per cycle.

^cMinus 0.5°C per cycle.