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Team:TU Darmstadt/Notebook/Methods/Restriction digest
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Revision as of 15:53, 16 October 2014
Restriction digestion
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.
