Team:Arizona State/Lab/Protocols/Restriction

Linearized Plasmid Backbone Restriction

• Preparation of Master mix:

• 5 uL NEB Buffer 4
• 0.5 uL BSA
• 0.5 uL EcoRI
• 0.5 uL PstI
• 0.5 uL DpnI
• 18 uL dH2O

• 4 uL linearized plasmid backbone + 4uL master mix in tube.
• Digest at 37C for 30 minutes. Heat shock at 80C for 20 minutes.

[http://openwetware.org/wiki/Endy:Restriction_Digest Open WetWare Restriction]

• Master mix (good for 8 restrictions)

• 40 uL Buffer 2
• 0.5 uL BSA
• 296 uL ddH2O

• 42.5 uL master mix + 300ng DNA + 1 uL of each restriction enzyme in tube.
• Digest at 37C for 1 hour (or longer). Heat shock at 80C for 20 minutes.