Difference between revisions of "EcoRI"

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(Notes)
(Notes)
 
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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)''':
  
==References==
+
==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]]

Latest revision as of 11:07, 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

  1. 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. PubMed ID:4343967 | HubMed [Morrow-PNAS-1972]
    Discusses cleavage of DNA at a unique location by EcoRI
  2. 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. PubMed ID:4343959 | HubMed [Mulder-PNAS-1972]
    Specificity of EcoRI
  3. 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. PubMed ID:4343974 | HubMed [Hedgpeth-Proc-Natl-Acad-Sci-USA-1972]
    Identifies the overhang sequence produced by EcoRI
  4. Bigger CH, Murray K, and Murray NE. Recognition sequence of a restriction enzyme. Nat New Biol. 1973 Jul 4;244(131):7-10. PubMed ID:4578426 | HubMed [Bigger-Nat-New-Biol-1973]
    No abstract available online.
  5. 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. PubMed ID:4343968 | HubMed [Mertz-Proc-Natl-Acad-Sci-USA-1972]
    Use of EcoRI to generate DNA fragments that can be ligated
  6. 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. PubMed ID:4594039 | HubMed [Cohen-PNAS-1973]
    Use of EcoRI to generate recombinant DNA fragments
All Medline abstracts: PubMed | HubMed