Team:TU Darmstadt/protocols/Restriction digest


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Restriction digest

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

Singel DNA Digestion (20 µL batch)
The following is an example of a typical analytical single restriction enzyme digestion: Add up the following:
14 µL nuclease-free water
2 µL 10x NEBuffer
1 µL Restriction Enzyme (10 u)
1 µL DNA Sample (1µg)
1 µL Restriction Enzyme (10 u)

Incubate for 1 h at an temperatur of 37°C
inactivate enzymes by incubating at 65°C for 20 min
Use 1µl of the sample and add 5µl of Blue/Orange 6X Loading Dye to proceed a gel analysis.
Larger scale restriction enzyme digestions can be accomplished by scaling this basic reaction proportionately.

Multiple Restriction Enzyme Digests (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 still works good for every used enzyme) Following the single restriction enzyme digestion by using 1 µL of the additional enzyme and take off 1 µL from the nuclease-free water.