Team:XJTLU-CHINA/Protocols Digestion
Digestion (NEB enzymes)
- Single-enzyme digestion
- Add 100-1000 ng DNA into an empty 1.5 ml microcentrifuge tube.
- Add 5μl 10×NEB buffer (dependent on enzymes) to the tube.
- Complement ddH2O to a total volume of 49 μl system.
- At last, add 1μl restriction enzyme and incubate the reaction system at proper temperature (normally 37 ℃) for 1 hour. The digestion efficiency can be increased through a prolonged incubation time.
- Double-enzyme digest
Reagents:
Usually the volume of reaction can vary:
Procedure:
(The work conditions of corresponding restriction enzyme can be referenced on NEB official website)
Application in this project: pNZ8148 plasmid digestion by PstI
Incubate at 37℃ for 1 hour. Heat kill at 80℃ for 20 minutes.
(0.5 μl BSA can be added in reaction to increase digest efficiency, and buffer should be decided depending on the enzymes used to maximize the cutting efficiency)
Applications in this project: PstI and EcoRI for Linearized pSB1C3, PstI and EcoRI for BBa_2309022, BBa_2309023 and BBa_2309027.
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