Component	Amount
10 × PfuTurbo Cx reaction buffer	5 μL
dNTP Mix (10 mM each dNTP)	1 μL
5hmC testprimer-F (10 μM)	1 μL
5hmC test primer-R (10 μM)	1 μL (see Note 23)
Bisulfite-treated DNA (from step 1 in Subheading 3.5.1)	2–4 μL (see Note 24)
PfuTurbo Cx Hotstart DNA polymerase (2.5 U/μL)	1 μL (see Note 25)
Milli-Q water	To a final volume of 50 μl