Amount (μl)
Component	Tube 1	Tube 2	Final concentration
Reaction buffer (2×)	25	25	1×
5-hmC dNTP mix (10 mM each)	1	1	200 μM each
5-hmC_F_1 (10 μM)	2	—	0.4 μM
5-hmC_R_1 (10 μM)	2	—	0.4 μM
5-hmC_F_ 2 (10 μM)	—	2	0.4 μM
5-hmC_R_ 2 (10 μM)	—	2	0.4 μM
pUC 19 vector (1 ng μl−1)	1		1 ng per 50 μl
ZymoTaq DNA Polymerase (5 U μl−1)	0.4	0.4	2 U per 50 μl PCR
Milli-Q water	to 50	to 50
Final volume	50	50