Table 3. PCR mix for 1st and 2nd PCR.

PCR step	Reagent	Concentration	1x	96x + 10%
1st PCR	Nuclease -free water	-	32.16	3376.80
	PCR Buffer	[10x]	4.00	420.00
	Forward primer mix	[50 μM each]	0.13	13.65
	Reverse primer mix	[50 μM each]	0.13	13.65
	dNTPs	[25 mM each]	0.40	42.00
	HotStar Taq	[5 U/μl]	0.18	18.90
	subtotal mix		37.00	3885.00
	cDNA template		3.00
	Total		40.00
2nd PCR	Nuclease-free water	-	31.66	3324.30
	PCR Buffer	[10x]	4.00	420.00
	Forward primer mix	[50 μM each]	0.13	13.65
	Reverse primer mix	[50 μM each]	0.13	13.65
	dNTPs	[25 mM each]	0.40	42.00
	HotStar Taq	[5 U/μl]	0.18	18.90
	subtotal mix		36.50	3832.50
	DNA template		3.50
	Total		40.00