From 2008.igem.org

Latest revision as of 12:52, 1 July 2008

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The protocols we used

• LB medium preparation
• Plasmid resuspension from IGEM paper spots
• Transformation
• Plasmid extraction
• BioBrick digestion with restriction enzymes
• DNA gel extraction
• DNA precipitation with sodium acetate
• Antarctic Phosphatase
• Ligation
• PCR

BioBrick digestion with restriction enzymes

(estimated time: 3 hours)

Materials needed:

• Roche restriction enzymes thawed on ice
• Roche buffer H
• Pre-warmed at 37°C bath
• Cut and gel-extracted vector
• ddH2O

• To open vectors:
  • a volume containing 1 µg of purified plasmid
  • 2 µl of buffer H
  • 1 µl of first enzyme
  • 1 µl of second enzyme
  • 20 µl final volume
  • incubate at 37°C for 3 hours
• To excide fragments:
  • 20 µl of purified plasmid when <7 µg have been extracted. A volume containing 7 µg otherwise.
  • 2.5 µl of buffer H
  • 1 µl of first enzyme
  • 1 µl of second enzyme
  • 25 µl final volume
  • incubate at 37°C for 2 hours and 30 minutes

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