Non disponible en dehors du Royaume-Uni et de l'Irlande

Analysis Note

Activity in PCR buffer: Not tested

Compatible endsSpe I ends are compatible with ends generated by Bln I, Nhe I, and Xba I.IsoschizomersThe enzyme is an isoschizomer of Bcu I and Ahl I.Methylation sensitivityAs 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°CPFGE testedSpe 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 assaySpe 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.

SuRE/Cut Buffer SystemThe buffer in bold is recommended for optimal activity A: 75-100% B: 75-100% H: 100% L: 75-100% M: 100%

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

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 activities1µg 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 activityApproximately 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.

Specificity

Recognition sites: *A*CTAGT*A*CTAGTRestriction site: *A↓*CTAGT*A↓*CTAGTHeat inactivation: Spe I can be heat inactivated by incubation at 65 °C for 15 minutes (up to 100 U/µg DNA).

Unit Definition

One unit is the enzyme activity that completely cleaves 1 µg Ad2 DNA in one hour at +37 °C in a total volume of 25 µl SuRE/Cut Buffer H.