Team:UNIPV-Pavia/Protocols/Digestion

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