Restriction digestion

Materials required

• Eppendorf tubes
• Pipettes
• Appropriate DNA/plasmid
• Appropriate restriction enzymes
• 10X buffer
• Water

Procedures

• Make up a 20 µl reaction mix as mentioned below:

1. 15 µl of DNA/plasmid
2. 1 µl of restriction enzyme 1
3. 1 µl of restriction enzyme 2
4. 2 µl of 10X buffer
5. 1 µl of water

• Incubate at 37°C for 3 hours

Notes

• No more than 10% of enzyme should be used for a single reaction - glycerol inhibits reaction
• 10x buffer must be diluted to 1x i.e. 10% final volume