Storage: −20°C
UNSPSC Code: 12352200
Components: Enzyme Solution; SuRE/Cut Buffer H 10x concentrated
RIDADR: NONH for all modes of transport
Analysis Note:
SuRE/Cut Buffer System
The buffer in bold is recommended for optimal activity
• A: 100%
• B: 100%
• H: 100%
• L: 25-50%
• M: 50-75%
Application:
EcoR I from Escherichia coli BS5 has been used to digest bacterial DNA.
General description:
EcoR I recognizes the sequence G↓*A*ATT*C and generates fragments with 5′-cohesive termini. EcoR I is inhibited if N6-methyladenine occurs at either or both A residues (*) in the recognition site; it is also inhibited if 5-methylcytosine occurs at the site indicated (*). EcoR I is an isoschizomer to Rsr I.
Other Notes:
For life science research only. Not for use in diagnostic procedures.
Compatibility:
EcoR I generates ends that are compatible with fragments generated by Acs I and Mun I.
DNA Profile:
Number of cleavage sites on different DNAs
• λ: 5
• φX174: 0
• Ad2: 5
• M13mp7: 2
• pBR322: 1
• pBR328: 1
• pUC18: 1
• SV40: 1
Preparation Note:
Quality
Absence of nonspecific endonuclease activities
1 μg λDNA is incubated for 16 hours in 50 μl SuRE/Cut Buffer H with an excess of EcoR I. 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 I 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.
Specificity:
Star Activity
The enzyme shows star activity under non-optimal conditions. The recognition specificity of EcoR I is altered by addition of increasing amounts of hydrophobic reagents and glycerol to the incubation mixture. The relaxed specificities of EcoR I are N/AATTN or RRATYY. When digesting genomic DNA with high concentrated EcoR I note that a 1:10 dilution of enzyme solution is the minimum dilution.
Recognition sites: G*A*ATT*C
G*A*ATT*C
Restriction site: G↓*A*ATT*C
G↓*A*ATT*C
Heat inactivation: EcoR I can be heat inactivated by incubation at 65 °C for 15 minutes (up to 10 U/μg DNA).
Star activity: To avoid star activity, always use the optimal buffer system and enzyme amount recommended in the package insert. Make sure that the DNA preparation is free of organic solvents and contaminating salts.