Engineering BioBrick vectors from BioBrick parts/Restriction digest

Materials

• Restriction enzymes (EcoRI, SpeI, XbaI or PstI) from NEB
• Bovine Serum Albumin (BSA)
• Deionized, sterile H₂O
• 0.5-1 μg DNA
• Sterile 0.6mL plastic tubes

Equipment

• DNA Engine Peltier Thermal Cycler (PTC-200) from MJ Research, Inc. (now Bio-Rad Laboratories, Inc., Hercules, CA).

Digest mix

• 1X NEB2 buffer
• 100 μg/mL BSA
• 1 μL BioBrick enzyme 1
• 1 μL BioBrick enzyme 2

deionized, sterile H₂O to 50 μL

Procedure

Vortex all reagents before use.

1. Add appropriate amount of deionized H₂O to sterile 0.6 mL tube
2. Add restriction enzyme buffer.
3. Add BSA.
4. Add DNA.
5. Add each enzyme.
   Also, the enzyme is in some percentage of glycerol which tends to stick to the sides of your tip. To ensure you add only 1 μL, just touch your tip to the surface of the liquid when pipetting.
6. Incubate for 2 hours at 37°C.
7. Incubate for 20 mins at 80°C to heat inactivate enzyme.
   This step is sufficient to inactivate even Pst I.
8. Incubate 4°C until you pull the reaction out of the thermal cycler.