Engineering BioBrick vectors from BioBrick parts/Restriction digest
< Engineering BioBrick vectors from BioBrick parts
- Restriction enzymes (EcoRI, SpeI, XbaI or PstI) from NEB
- Bovine Serum Albumin (BSA)
- Deionized, sterile H2O
- 0.5-1 μg DNA
We performed all restriction digests by mixing 0.5-1 $\mu$g DNA, 1X NEBuffer 2, 100 $\mu$g/ml Bovine Serum Albumin, and 1 $\mu$L each needed restriction enzyme in a 50 $\mu$L total volume.
- DNA Engine Peltier Thermal Cycler (PTC-200) from MJ Research, Inc. (now Bio-Rad Laboratories, Inc., Hercules, CA).
- 1X NEB2 buffer
- 100 μg/mL BSA
- 1 μL BioBrick enzyme 1
- 1 μL BioBrick enzyme 2
deionized, sterile H2O to 50 μL
- Add appropriate amount of deionized H2O to sterile 0.6 mL tube
- Add restriction enzyme buffer.
Vortex buffer before pipetting to ensure that it is well-mixed.
- Add BSA.
Vortex BSA before pipetting to ensure that it is well-mixed.
- Add DNA.
Vortex DNA before pipetting to ensure that it is well-mixed.
- Add each enzyme.
Vortex enzyme before pipetting to ensure that it is well-mixed.
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.
- Incubate for 2 hours at 37°C.
- Incubate for 20 mins at 80°C to heat inactivate enzyme.
This step is sufficient to inactivate even Pst I.
- Incubate 4°C until you pull the reaction out of the thermal cycler.