Restriction/Digestion

Materials:

• 25 µL DNA (but varies depending on your DNA conc.)
• 10 µL FD-buffer
• 1 µL restriction enzyme (1 & 2) (you can also use 2.5µL of each RE, which showed good results in previous digestions (AJ+PF))
• Using 1µL RE: 63ul ddH2O. Using 2.5µL RE: 60 µL ddH2O (Total volume of 100 µL)

1. Turn the thermo block on 30 min before use. Mix everything – add the enzymes last. Leave for 2 hour at 37 C in thermo block/incubator.
2. To deactivate the enzyme leave it at 65 C in a thermo block for 10 min.
3. Keep on ice until its cooled down.

IMPORTANT: If you only use one RE on your plasmid SAP treatment is required:

• Mix 50µL of the restricted DNA with 2.5µL SAP and 5µL SAP-buffer
• Put it in the incubator 37°C for 60 min
• 15 minutes on heat block 65°C to deactivate enzyme
• Place on ice

Enzyme selection for BioBricks digest A BioBricks part:

 ---E--X---Part---S--P--- 

Vector with upstream part - S,P Downstream Insert - X,P Upstream Insert - E,S Vector with downstream part - E,X

E= EcoRI X= XbaI S= SpeI P= PstI

Enzymes should be kept in the icebox from the freezer at all times.