Team:British Columbia/Protocols/Restriction Digests

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British Columbia - 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.

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.