From 2007.igem.org

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• Applications:
  1. Identification of insert presence

• Time to complete protocol:
  • Lab time: 15min, 15min.
  • Waiting time: 1-3hours
• Approximate cost of materials: $0.00

Method from primary and secondary reagents

Primary & secondary Reagents Required including controls

• Melbourne/primary Restriction enzymes and buffer
• DNA for digestion
• milliQ water

Method including controls

For 20uL reation volume

1. Make the following reaction mixture on ice.
   • Reaction Mixture
     • 0.5uL Enzyme 1
     • 0.5uL Enzyme 2
       • Add enzymes last only take out of the freezer once ready to add.
     • 2uL appropriate 10x buffer (see table on fridge)
     • 2uL 10x BSA
     • 10uL MilliQ
     • 5uL DNA
       • If performing the same digest for multiple DNA samples make up mastermix of buffer enzyme soultion and make 15uL aliquots to which to add the DNA.
       • Volume DNA may vary depending on concentration, vary water volume to achieve 20uL reaction volume.
2. Incubate for 1-3 hrs at 37 degrees.
3. Stop reaction with addition of 5uL 6x DNA loading dye.
4. Can be stored at -20 or run on gel immediately.

Equipement Required

• microfuge tubes
• Ice box and ice
• 37 degree incubator
• Pipettes and tips

References