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 &mu;g 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
deionized, sterile H2O to 50 &mu;L
 * 1X NEB2 buffer
 * 100 &mu;g/mL BSA
 * 1 &mu;L BioBrick enzyme 1
 * 1 &mu;L BioBrick enzyme 2

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 &mu;L, just touch your tip to the surface of the liquid when pipetting.
 * 6) Incubate for 2 hours at 37&deg;C.
 * 7) Incubate for 20 mins at 80&deg;C to heat inactivate enzyme. This step is sufficient to inactivate even Pst I.
 * 8) Incubate 4&deg;C until you pull the reaction out of the thermal cycler.