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Team:Cambridge/Protocols/Restriction Enzyme Digestion
From 2011.igem.org
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:# perform gel electrophoresis (10μl of DNA-restriction enzyme mixture in each well) | :# perform gel electrophoresis (10μl of DNA-restriction enzyme mixture in each well) | ||
:# compare with predicted fragment sizes | :# compare with predicted fragment sizes | ||
| + | :# remember about molecular weight markers | ||
===Safety=== | ===Safety=== | ||
Revision as of 14:47, 15 July 2011
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Restriction Enzyme Digestion
A method that allows to create a restriction map of a given DNA fragment. Widely used to test for the correct integration of a cloned sequence into a vector.
Theory
How it works
Practice
- Master mix preparation
- 2.0μl of DNA
- 0.5μl of each restriction enzyme (or the respective amount of water for the control with uncut plasmids)
- 0.1μl of BSA - acetylated Bovine Serum Albumin enhances the performance of restriction enzymes
- 5.9μl of water
- 1.0μl of 2×NEBuffer
- Check for the compatibility of restriction enzymes chosen
To check if the two selected restriction enzymes can perform effective catalysis in the same solution, go to the website of [http://www.neb.com New England Biolabs] → select [http://www.neb.com/nebecomm/DoubleDigestCalculator.asp? Double Digest Finder] → choose which restriction enzymes you want to use → follow the digest recommendations.
- Procedure
- incubate at 37°C for 2 hours
- perform gel electrophoresis (10μl of DNA-restriction enzyme mixture in each well)
- compare with predicted fragment sizes
- remember about molecular weight markers
Safety
The safety implication of the procedure.
