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Restriction Endonucleases >
Restriction Endonucleases >
EcoRI |
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Recognition Site:
isoschizomers | compatible ends | single letter code
Source:
An E. coli strain that carries the cloned EcoRI gene from E. coli RY13 (R.N. Yoshimori).
Reagents Supplied:
NEBuffer EcoRI (10X)
Enzyme Properties
Activity in NEBuffers:
| NEBuffer 1: | | 100% | | NEBuffer 2: | | 100% | | NEBuffer 3: | | 100% | | NEBuffer 4: | | 100% |
When using a buffer other than the optimal (supplied) NEBuffer, it may be necessary to add more enzyme to achieve complete digestion.
Methylation Sensitivity:
| dam methylation: Not sensitive | | dcm methylation: Not sensitive | | CpG methylation: Impaired by overlapping |
Heat Inactivation:
65°C for 20 minutes
Survival in a Reaction:
Minimum units to digest 1 µg of substrate DNA in 16 hours: 0.13 unit(s)
Reaction & Storage Conditions
Reaction Conditions:
1X NEBuffer EcoRI
Incubate at
37°C.
1X NEBuffer EcoRI:
100 mM Tris-HCl
50 mM NaCl
10 mM MgCl2
0.025 % Triton X-100
pH 7.5 @ 25°C
Unit Definition:
One unit is defined as the amount of enzyme required to digest 1 µg of λ DNA in 1 hour at 37°C in a total reaction volume of 50 µl.
Concentration:
20,000 units/ml and 100,000 units/ml
Unit Assay Substrate:
λ DNA
Storage Conditions:
300 mM NaCl
10 mM 2-Mercaptoethanol
0.1 mM EDTA
200 µg/ml BSA
50% Glycerol
0.15% Triton X-100
pH 7.5 @ 25°C
Storage Temperature:
-20°C
Diluent Compatibility:
Diluent C
Notes
General notes:- Conditions of low ionic strength, high enzyme concentration, glycerol concentration > 5%, or pH > 8.0 may result in star activity.
FAQs
- What is Star Activity and how can it be avoided?
Quality Control for Current Lot
Quality control values for a specific lot can be found on the datacard which accompanies each product.
Ligation and Re-cutting:
After a 100-fold overdigestion with EcoRI, > 95%
of the DNA fragments can be ligated with T4 DNA Ligase (at a 5' termini concentration of 1-2 μM)
at 16ºC. Of these ligated fragments, > 95% can be recut with EcoRI.
16-Hour Incubation:
A 50 μl reaction containing 1 μg of λDNA
and 100 units of EcoRI incubated for 16 hours at 37ºC
resulted in a DNA pattern free of detectable nuclease degradation as determined by agarose gel electrophoresis.
Exonuclease Activity:
Incubation of a 50 μl reaction containing 200 units of EcoRI with 1 μg of a mixture of single and double-stranded
[3H] E. coli DNA (105 cpm/μg) for 4 hours at 37ºC
released < 0.1% of the total radioactivity.
Blue/White Screening Assay:
An appropriate vector is digested at a unique site within the
lacZα gene with a 10-fold excess of enzyme.
The vector DNA is then ligated, transformed, and plated onto Xgal/IPTG/Amp plates.
Successful expression of β-galactosidase is a function of how intact its gene remains after cloning,
an intact gene gives rise to a blue colony, removal of even a single base gives rise to a white colony.
Enzyme preparations must produce fewer than 3% white colonies to be Blue/White certified.
Reagents Sold Separately
NEBuffer EcoRI
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