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From 2012.igem.org

Restriction Digests

1. Prepare reaction mix, following the basic ratios below. Each reaction requires at least 4 uL of master mix.

Master Mix

Ratio	Name of Reagent	Quantity/reaction (uL)
5x	NEB Buffer 2	1
0.5x	BSA	0.1
0.5x	Dpn1	0.1
0.5x	Eco-RI (HF)	0.1
0.5x	Pst1	0.1
18x	dH2O	3.6
TOTAL		5 uL

Notes:

• do not add Dpn1 when working with plasmid DNA - it will cut all methylated DNA.
• keep the restriction enzymes as close to -20°C as possible for as long as possible.

2. Add 4 uL of master mix and 4 uL of undigested template DNA (plasmid or PCR product) to a 200 uL PCR tube.

3. Heat the mixtures at 37°C for 30 minutes for optimal enzyme activity, then 80°C for 20 minutes to inactivate the enzymes.

• easiest to use a thermocycler

4. Follow up with ligation or store products at -20°C.