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Team:UNIPV-Pavia/Protocols/Digestion
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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
