Component	Volume (µl)
	1 reaction	5 reactions	10 reactions
HF buffer (5 ×)	4.00	20.0	40.0
dNTPs (10 mM)	0.40	2.0	4.0
DMSO, 100%	0.60	3.0	6.0
F primer (100 µM)	0.05	0.25	0.50
R primer (100 µM)	0.05	0.25	0.50
DNA fragments*	x + y + (z)	5[x + y + (z)]	10[x + y + (z)]
Ultrapure water	14.7 – [x + y + (z)]	73.5 – 5[x + y + (z)]	147 – 10[x + y + (z)]
Phusion® DNA polymerase	0.20	1.0	2.0
Total volume	20.00	100.0	200.0

^*

Add a volume that provides 2–5 ng of DNA fragment per reaction. These master mixes can be used for two (x, y) or three (x, y, z) fragment assemblies.