EcoRI: Difference between revisions
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* '''Reaction Volumes''' - I used to have very inconsistent results when digesting with EcoRI, using the 20μl reaction volume. Using the Knight Lab's 50μl mix has recently given good results. I haven't tested this enough to say it is a statistically significant result. --[[User:Bcanton|BC]] 13:39, 2 Jun 2005 (EDT) | * '''Reaction Volumes''' - I used to have very inconsistent results when digesting with EcoRI, using the 20μl reaction volume. Using the Knight Lab's 50μl mix has recently given good results. I haven't tested this enough to say it is a statistically significant result. --[[User:Bcanton|BC]] 13:39, 2 Jun 2005 (EDT) | ||
* The custom EcoRI buffer provided by NEB contains Triton-X100. This detergent is very inhibitory in transformations, so direct ligation and cloning of DNA cut in this buffer is problematic. Remarkably small amounts of this buffer dramatically reduces transformation efficiency. | * The custom EcoRI buffer provided by NEB contains Triton-X100. This detergent is very inhibitory in transformations, so direct ligation and cloning of DNA cut in this buffer is problematic. Remarkably small amounts of this buffer dramatically reduces transformation efficiency. | ||
* The FastDigest restriction enzymes from Fermentas all work very efficiently in one universal buffer. I perform digests in a 30 μL reaction volume. *'''[[User:Karmella Haynes|Karmella Haynes]] 14:07, 19 January 2012 (EST)''': | |||
== | ==External links== | ||
[http://www.neb.com/nebecomm/products/productR0101.asp EcoRI from NEB]<br /> | [http://www.neb.com/nebecomm/products/productR0101.asp EcoRI from NEB]<br /> | ||
[http://www.promega.com/catalog/catalogproducts.asp?catalog_name=Promega_Products&category_name=EcoR+I&cookie%5Ftest=1 EcoRI from Promega]<br /> | [http://www.promega.com/catalog/catalogproducts.asp?catalog_name=Promega_Products&category_name=EcoR+I&cookie%5Ftest=1 EcoRI from Promega]<br /> | ||
==References== | |||
<biblio> | |||
#Morrow-PNAS-1972 pmid=4343967 | |||
// Discusses cleavage of DNA at a unique location by EcoRI | |||
#Mulder-PNAS-1972 pmid=4343959 | |||
// Specificity of EcoRI | |||
#Hedgpeth-Proc-Natl-Acad-Sci-USA-1972 pmid=4343974 | |||
// Identifies the overhang sequence produced by EcoRI | |||
#Bigger-Nat-New-Biol-1973 pmid=4578426 | |||
// No abstract available online. | |||
#Mertz-Proc-Natl-Acad-Sci-USA-1972 pmid=4343968 | |||
// Use of EcoRI to generate DNA fragments that can be ligated | |||
#Cohen-PNAS-1973 pmid=4594039 | |||
// Use of EcoRI to generate recombinant DNA fragments | |||
</biblio> | |||
[[Category:Material]] [[Category:Enzyme]] [[Category:Endonuclease]] | [[Category:Material]] [[Category:Enzyme]] [[Category:Endonuclease]] | ||
Latest revision as of 12:07, 19 January 2012
Properties
Recognition site
http://www.neb.com/nebecomm/productfiles/314/images/EcoR-I-cutsite_1.gif
Buffers
NEBuffer EcoR I
Notes
- Improving the efficiency of EcoRI/SpeI Double Digest.
- Reaction Volumes - I used to have very inconsistent results when digesting with EcoRI, using the 20μl reaction volume. Using the Knight Lab's 50μl mix has recently given good results. I haven't tested this enough to say it is a statistically significant result. --BC 13:39, 2 Jun 2005 (EDT)
- The custom EcoRI buffer provided by NEB contains Triton-X100. This detergent is very inhibitory in transformations, so direct ligation and cloning of DNA cut in this buffer is problematic. Remarkably small amounts of this buffer dramatically reduces transformation efficiency.
- The FastDigest restriction enzymes from Fermentas all work very efficiently in one universal buffer. I perform digests in a 30 μL reaction volume. *Karmella Haynes 14:07, 19 January 2012 (EST):
External links
EcoRI from NEB
EcoRI from Promega
References
- Morrow JF and Berg P. Cleavage of Simian virus 40 DNA at a unique site by a bacterial restriction enzyme. Proc Natl Acad Sci U S A. 1972 Nov;69(11):3365-9. DOI:10.1073/pnas.69.11.3365 |
Discusses cleavage of DNA at a unique location by EcoRI
- Mulder C and Delius H. Specificity of the break produced by restricting endonuclease R 1 in Simian virus 40 DNA, as revealed by partial denaturation mapping. Proc Natl Acad Sci U S A. 1972 Nov;69(11):3215-9. DOI:10.1073/pnas.69.11.3215 |
Specificity of EcoRI
- Hedgpeth J, Goodman HM, and Boyer HW. DNA nucleotide sequence restricted by the RI endonuclease. Proc Natl Acad Sci U S A. 1972 Nov;69(11):3448-52. DOI:10.1073/pnas.69.11.3448 |
Identifies the overhang sequence produced by EcoRI
- Bigger CH, Murray K, and Murray NE. Recognition sequence of a restriction enzyme. Nat New Biol. 1973 Jul 4;244(131):7-10. DOI:10.1038/newbio244007a0 |
No abstract available online.
- Mertz JE and Davis RW. Cleavage of DNA by R 1 restriction endonuclease generates cohesive ends. Proc Natl Acad Sci U S A. 1972 Nov;69(11):3370-4. DOI:10.1073/pnas.69.11.3370 |
Use of EcoRI to generate DNA fragments that can be ligated
- Cohen SN, Chang AC, Boyer HW, and Helling RB. Construction of biologically functional bacterial plasmids in vitro. Proc Natl Acad Sci U S A. 1973 Nov;70(11):3240-4. DOI:10.1073/pnas.70.11.3240 |
Use of EcoRI to generate recombinant DNA fragments
