Spe I

Кат. номер

11008951001

11008943001

11207644001

Spe I recognizes the sequence *A*CTAGT and generates fragments with 5-cohesive termini.
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).
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

• : 0
• X174: 0
• Ad2: 3
• M13mp7: 0
• pBR322: 0
• pBR328: 0
• pUC18: 0
• SV40: 0

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.
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 ( mAm 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 x Spe I fragments.
SuRE/Cut Buffer System
The buffer in bold is recommended for optimal activity

• A: 75-100%
• B: 75-100%
• H: 100%
• L: 75-100%
• M: 100%

Activity in PCR buffer: Not tested
For life science research only. Not for use in diagnostic procedures.

Quality Level	100,
form	solution
packaging	pkg of 1,000 U (11008951001 [10 U/µl]), pkg of 1,000 U (11207644001 [40 U/µl]), pkg of 200 U (11008943001 [10 U/µl])
Торговая марка	Roche
parameter	37 °C optimum reaction temp.
shipped in	dry ice
storage temp.	20°C

RIDADR

NONH for all modes of transport