Analysis Note

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

Activity in PCR buffer: Not tested

PFGE testedBln 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 10 U of enzyme/µg DNA and 4 hour incubation.

DNA Profile

Number of cleavage sites on different DNAs λ: 2 φX174: 0 Ad2: 2 M13mp7: 0 M13mp18:0 pBR322: 0 pBR328: 0 pUC18: 0 SV40: 2

General description

Bln I recognizes the sequence C↓CTAGG and generates fragments with 5′-cohesive termini.Compatible endsBln I ends are compatible with ends generated by Nhe I, Spe I and Xba I.IsoschizomersBln I is an isoschizomer of Avr II.Note: The complete 13 site Avr II restriction map of the E.coli genome has been reported.Methylation sensitivityThe enzyme is not known to be affected by methylation.

Other Notes

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

Quality

Absence of nonspecific endonuclease activities1 µg λDNA is incubated for 16 hours in 50 µl SuRE/Cut Buffer H with an excess of Bln 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 5 µg [³H] labeled calf thymus DNA are incubated with 3 µl Bln 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.Typical ligation and recutting assayBln I fragments obtained by complete digestion of 1 µg λ × EcoR I DNA ligated for 16 hours at +4°C with 1 U T4 DNA Ligase in 10 µl buffer that contains 66 mM Tris-HCl, 5 mM MgCl₂, 5 mM Dithiothreitol, 1 mM ATP, pH 7.5 (at +20°C). The percentages of product that can be ligated and subsequently recut with Bln I and EcoR I (yielding the typical pattern of λ × EcoR I × Bln I fragments) are stated under "Lig" and "Rec" in the certificate of analysis.

Specificity

Recognition sites: CCTAGGCCTAGGRestriction site: C↓CTAGGC↓CTAGGHeat inactivation: No inactivation of Bln I after incubation at 65 °C for 15 minutes.

Unit Definition

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