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Team:British Columbia/Protocols/Restriction Digests
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| + | =Restriction Digest Protocol= | ||
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| + | 1. Prepare reaction mix, following the basic ratios below. Each reaction requires at least 4 uL of master mix. | ||
| + | |||
| + | 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 | ||
Revision as of 00:33, 26 June 2012
Restriction Digest Protocol
1. Prepare reaction mix, following the basic ratios below. Each reaction requires at least 4 uL of master mix.
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
