Storage: −20°C
UNSPSC Code: 12352200
Components: Enzyme Solution; SuRE/Cut Buffer H 10x concentrated
RIDADR: NONH for all modes of transport
Analysis Note:
Compatible ends
Spe I ends are compatible with ends generated by Bln I, Nhe I, and Xba I.
Isoschizomers
The enzyme is an isoschizomer of Bcu I and Ahl I.
Methylation sensitivity
As indicated by (*) on the recognition sequence above, Spe I is inhibited by the presence of N6-methyladenine and 5′-methylcytosine ( mA↓m CTAGT).
Incubation temperature
+37°C
PFGE tested
Spe I has been tested in Pulsed-Field Gel Electrophoresis (on bacterial chromosomes). For cleavage of genomic DNA (E. coli C 600) embedded in agarose for PFGE analysis, we recommend using 10U of enzyme/μg DNA and 4 hour incubation time.
Ligation and recutting assay
Spe I fragments obtained by complete digestion of 1μg Ad2 DNA are ligated with 1U T4 DNA Ligase in a volume of 10μl by incubation for 16 hours at +4°C in 66mM Tris-HCl, 5mM MgCl2, 5mM Dithiothreitol, 1mM ATP, pH 7.5 (at +20°C), resulting in >90% recovery of Ad2 DNA.
Subsequent re-cutting with Spe I yields >95% of the typical pattern of Ad2 × Spe I fragments.
General description:
Spe I recognizes the sequence *A↓*CTAGT and generates fragments with 5′-cohesive termini.
Other Notes:
For life science research only. Not for use in diagnostic procedures.
Quality:
Absence of nonspecific endonuclease activities
1μg Ad2 DNA is incubated for 16 hours in 50μl SuRE/Cut Buffer H with an excess of Spe 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 5μg [3H] labeled calf thymus DNA are incubated with 3μl Spe I for 4 hours at +37°C in a total volume of 100μl 50mM Tris-HCl, 10mM MgCl2, 1mM Dithioerythritol, pH approximately 7.5. Under these conditions, no release of radioactivity is detectable, as stated in the certificate of analysis.
DNA Profile:
Number of cleavage sites on different DNAs
• λ: 0
• φX174: 0
• Ad2: 3
• M13mp7: 0
• pBR322: 0
• pBR328: 0
• pUC18: 0
• SV40: 0
Specificity:
Recognition sites: *A*CTAGT
*A*CTAGT
Restriction site: *A↓*CTAGT
*A↓*CTAGT
Heat inactivation: Spe I can be heat inactivated by incubation at 65 °C for 15 minutes (up to 100 U/μg DNA).