Short explanation:

In order to insert DNA fragments into plasmids via ligation it is necessary to digest both components with restriction enzymes.

Procedure: Single DNA Digestion (20 µL batch)

The following is an example of a typical single restriction enzyme digestion.

Mix:

- 14 µL nuclease-free water

- 2 µL 10x NEBuffer

- 1 µL Restriction Enzyme (10 u)

- 1 µL DNA Sample (1µg) 

Incubate for 30 min at 37°C.

Inactivate enzymes by incubating at 80°C for 20 min.

Use 5 µl of the sample and add 1 µl 6X Loading Dye to proceed a agarose gel electrophoresis.

Larger scale restriction enzyme digestions can be accomplished by scaling this basic reaction proportionately.

Procedure: Multiple Restriction Enzyme Digestion (20 µL batch)

Use the optimal buffer supplied with one enzyme if the activity of the second enzyme is acceptable in that same buffer. NEB Buffer 4 usually works well for every used enzyme. Follow the single restriction enzyme digestion by using 1 µL of the additional enzyme and take 1 µL less nuclease-free water.