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Restriction Digest Protocol

Protocol can be found at: http://parts.igem.org/Help:Protocols/Restriction_Digest

Materials

  -Ice and bucket/container
  -Part 1 DNA
  -Part 2 DNA
  -Linearized plasmid backbone (25ng/µl)
  -dH2O
  -NEB Buffer 2, 3.1,or Ecor1 
  -BSA (Not needed with 3.1 buffer)
  -Restriction Enzymes: EcoRI, SpeI, XbaI, PstI, DnpI
  -Thermal cycler

Procedure

1. Keep all enzymes and buffers used on ice.

2. Thaw NEB Buffer and BSA (if needed- NEB buffers with .1 do not require additional BSA ie. NEB buffer 3.1) in room temperature water.

3. Add 250ng of DNA to the appropriately labeled tube.

4. In the Part 1 tube: Add 0.5µL of EcoRI, and 0.5µL of SpeI.

5. In the Part 2 tube: Add 0.5µL of XbaI, and 0.5µL of PstI.

6. In the plasmid tube: Add 0.5µL of EcoRI, 0.5µL of PstI, and 0.5µL of Dpn1.

7. Add 0.5µL of BSA to each tube. (If required)

8. Add dH20 to a final volume of 20µL.

9. Preset thermal cycler program to run the restriction digests at 37°C for 30 minutes, then 80°C for 20 minutes.