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Restriction Digest protocol

Brown iGEM 2010, modified from Knight Lab protocol.

Materials

• Restriction enzymes (we use NEB)
• NEB buffer 2/3 (use buffer finder on NEB website. Buffer 2 is standard for iGEM enzymes)
• BSA
• Deionized, sterile H2O

Digest mix

The reaction below is a 50ul reaction, 100ul reactions are also common if you want your DNA to be dilute.

• 5ul NEB Buffer 2
• Xul DNA (usually ~500ng)
• 0.5ul 100x BSA
• 1ul Restriction Enzyme (regardless of the reaction, 1ul is used because this generally represents a 10-25 fold excess. It is also sticky due to its glycerol content, so pipetting can be difficult)
• 1ul Restriction Enzyme 2 (for double digests)
• (42.5-X)ul dH2O

Protocol

1. Add appropriate amount of deionized H2O to sterile 0.6 mL tube
2. Add restriction enzyme buffer to the tube.
   • Vortex buffer before pipetting to ensure that it is well-mixed.
3. Add BSA to the tube.
   • Vortex BSA before pipetting to ensure that it is well-mixed.
4. Add appropriate amount of DNA to be cut to the tube.
   • Vortex DNA before pipetting to ensure that it is well-mixed.
5. Add 1 μL of each enzyme.
   • Vortex enzyme before pipetting to ensure that it is well-mixed.
   • Also, the enzyme is in some percentage of glycerol which tends to stick to the sides of your tip. To ensure you add only 1 μL, just touch your tip to the surface of the liquid when pipetting.
6. Place in thermal cycler (MJ Research, PT-200) and run digest protocol.
   1. 4-6 hour incubation at 37°C
      • Use a longer incubation time if you have time or are worried about the efficiency of cutting. I think this time can be shortened to 2 hrs while still cutting to completion.
   2. 20 mins at 80°C to heat inactivate enzyme.
      • This step is sufficient to inactivate even Pst I.
   3. 4°C forever (or until you pull the reaction out of the thermal cycler).
7. Generally, use some method of DNA purification to eliminate enzymes and salt from the reaction.