Digestion DNA: Difference between revisions
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This enzymatic technique can be used for cleaving DNA molecules at specific sites, ensuring that all DNA fragments that contain a particular sequence have the same size; furthermore, each fragment that contains the desired sequence has the sequence located at exactly the same position within the fragment. The cleavage method makes use of an important class of DNA-cleaving enzymes isolated primarily from bacteria. These enzymes are called restriction endonucleases or restriction enzymes, and they are able to cleave DNA molecules at the positions at which particular short sequences of bases are present. | This enzymatic technique can be used for cleaving DNA molecules at specific sites, ensuring that all DNA fragments that contain a particular sequence have the same size; furthermore, each fragment that contains the desired sequence has the sequence located at exactly the same position within the fragment. The cleavage method makes use of an important class of DNA-cleaving enzymes isolated primarily from bacteria. These enzymes are called restriction endonucleases or restriction enzymes, and they are able to cleave DNA molecules at the positions at which particular short sequences of bases are present. | ||
== Procedure == | |||
=== Digestion === | |||
Total amount of plasmid DNA: ~10µg (less is also possible). | Total amount of plasmid DNA: ~10µg (less is also possible). | ||
Add: | Add: | ||
* 10µl enzyme buffer</li> | |||
* Enzyme: Unit ==>1 U digest 1µg in 1h at 37°C (~ 2 μl enzyme)</li> | |||
* 1 μl BSA (check manuals instructions of the enzyme if it’s necessary or not!)</li> | |||
* Fill up the reaction volume to 100 μl with dd water</li> | |||
* '''2-3 hours restriction at 37°C''' | |||
== References == | |||
* ''Hartl, Daniel L., Jones, Elizabeth W. (2001), Genetics: Analysis of Genes and Genomes, Fifth Edition'' | |||
Latest revision as of 10:45, 27 March 2015
This enzymatic technique can be used for cleaving DNA molecules at specific sites, ensuring that all DNA fragments that contain a particular sequence have the same size; furthermore, each fragment that contains the desired sequence has the sequence located at exactly the same position within the fragment. The cleavage method makes use of an important class of DNA-cleaving enzymes isolated primarily from bacteria. These enzymes are called restriction endonucleases or restriction enzymes, and they are able to cleave DNA molecules at the positions at which particular short sequences of bases are present.
Procedure
Digestion
Total amount of plasmid DNA: ~10µg (less is also possible). Add:
- 10µl enzyme buffer
- Enzyme: Unit ==>1 U digest 1µg in 1h at 37°C (~ 2 μl enzyme)
- 1 μl BSA (check manuals instructions of the enzyme if it’s necessary or not!)
- Fill up the reaction volume to 100 μl with dd water
- 2-3 hours restriction at 37°C
References
- Hartl, Daniel L., Jones, Elizabeth W. (2001), Genetics: Analysis of Genes and Genomes, Fifth Edition