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ECORV-RO Roche

EcoR V

from Escherichia coli J62 pLG74

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Properties

Quality Level   100
form   solution
packaging   pkg of 10,000 U (10667153001 [10 U/μl])
  pkg of 10,000 U (11040197001 [40 U/μl])
  pkg of 2,000 U (10667145001 [10 U/μl])
mfr. no.   Roche
parameter   37 °C optimum reaction temp.
shipped in   dry ice
storage temp.   −20°C

Description

General description

EcoR V recognizes the sequence G*AT↓AT°C and generates fragments with blunt ends. EcoR V is inhibited by the presence of N6-methyladenine at the site (*) indicated on the recognition sequence. EcoR V is not inhibited by the presence of 5-methylcytosine at the indicated site (°) or 5-hydroxymethylcytosine. EcoR V is an isoschizomer of Eco32 I.

Specificity

Star Activity
EcoR V exhibits star activity under non-optimal conditions.
Recognition sites: G*ATAT °C
G*ATAT °C
Restriction site: G*AT↓AT °C
G*AT↓AT °C
Heat inactivation: No inactivation of EcoR V after incubation at 65 °C for 15 minutes.

Quality

Absence of nonspecific endonuclease activities
1 μg λDNA is incubated for 16 hours in 50 μl SuRE/Cut Buffer B with an excess of EcoR V. The number of enzyme units which do not change the enzyme-specific pattern is stated in the certificate of analysis.

Absence of exonuclease activity
Approximately 5 μg [3H] labeled calf thymus DNA are incubated with 3 μl EcoR V for 4 hours at +37°C in a total volume of 100 μl 50 mM Tris-HCl, 10 mM MgCl2, 1 mM Dithioerythritol, pH approximately 7.5. Under these conditions, no release of radioactivity is detectable, as stated in the certificate of analysis.

Compatibility

EcoR V generates fragments which are compatible with any blunt end.

DNA Profile

Number of cleavage sites on different DNAs
• λ: 21
• φX174: 0
• Ad2: 9
• M13mp7: 0
• pBR322: 1
• pBR328: 1
• pUC18: 0
• SV40: 1

Unit Definition

One unit is the enzyme activity that completely cleaves 1 μg λDNA in one hour at +37 °C in a total volume of 25 μl (1x) SuRE/Cut buffer B.

Preparation Note

Ligation and recutting assay
EcoR V fragments obtained by complete digestion of 1 μg λDNA are ligated with 1 U T4 DNA Ligase in a volume of 10 μl by incubation for 16 hours at +25°C in 66 mM Tris-HCl, 5 mM MgCl2, 5 mM Dithiothreitol, 1 mM ATP, pH 7.5 (at +20°C) resulting in >85% recovery of 1 μg λDNA × EcoR V fragments.
Subsequent re-cutting with EcoR V yields >95% of the typical pattern of λDNA × EcoR V fragments.

Analysis Note

SuRE/Cut Buffer System
The buffer in bold is recommended for optimal activity
• A: 25-50%
• B: 100%
• H: 50-75%
• L: 0-10%
• M: 25-50%

Activity in PCR buffer: 10%

Relative activity in PCR mix (Taq DNA Polymerase buffer) is 10%. The PCR mix contained λDNA, primers, 10 mM Tris-HCl (pH 8.3, 20 °C), 50 mM KCl, 1.5 mM MgCl2, 200 μM dNTPs, 2.5 U Taq DNA polymerase. The mix was subjected to 25 amplification cycles.Acvtivity in rection buffer of Pwo SuperYield DNA Polymerase PCR Mix is 25%. When supplemented with GC-RICH Solution activity remains at 25%. Pwo SuperYield DNA Polymerase PCR Mix is not available in U.S.

Other Notes

For life science research only. Not for use in diagnostic procedures.

Kit component only

Description

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Enzyme Solution    
SuRE/Cut Buffer B 10x concentrated    
Safety & Documentation

Safety Information

RIDADR 
NONH for all modes of transport

Documents

Certificate of Analysis (COA)

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Protocols & Articles
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