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Team:British Columbia/Protocols/Restriction Digests
From 2012.igem.org
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3. Heat the mixtures at 37°C for 30 minutes for optimal enzyme activity, then 80°C for 20 minutes to inactivate the enzymes. | 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 | *easiest to use a thermocycler | ||
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| + | 4. Follow up with ligation or store products at -20°C. | ||
Revision as of 00:49, 26 June 2012
Restriction Digests
1. Prepare reaction mix, following the basic ratios below. Each reaction requires at least 4 uL of 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.
