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Analysis Note

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

Absence of nonspecific endonuclease activities1µg λDNA is incubated for 16 hours in 50 µl SuRE/Cut Buffer H with an excess of Xba 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 3µl Xba 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.

Activity in PCR buffer: 60%Relative activity in PCR mix (Taq DNA Polymerase buffer) is 60%. The PCR mix contained λDNA, primers, 10 mM Tris-HCl (pH 8.3, 20 °C), 50 mM KCl, 1.5 mM MgCl₂, 200 µM dNTPs, 2.5 U Taq DNA polymerase. The mix was subjected to 25 amplification cycles.Activity in reaction buffer of Pwo SuperYield DNA Polymerase PCR Mix is 25%. When supplemented with GC-RICH Solution, activity increases to 100%. Pwo SuperYield DNA Polymerase PCR Mix is not available in U.S.

Application

The restriction enzyme Xba I has been used for the digestion of genomic DNA.

DNA Profile

Number of cleavage sites on different DNAs λ: 1 φX174: 0 Ad2: 5 M13mp7: 0 pBR322: 0 pBR328: 0 pUC18: 1 SV40: 0

General description

Xba I recognizes the sequence T↓*CTAGA and generates fragments with 5′-cohesive termini. The enzyme needs at least two nucleotides around the target sequence before it will cut the cleavage site.Compatible endsXba I ends are compatible with fragments generated by Avr I, Nhe I, and Spe I.IsoschizomersThe enzyme is not known to have isoschizomers.Methylation sensitivityXba I digestion of DNA is inhibited by the dam gene product of E. coli, which methylates the 6N position of adenine in the sequence GATC. The enzyme is also inhibited by 5-methylcytosine or 5-hydroxymethylcytosine at the site (*) indicated on the recognition sequence.Activity in SuRE/Cut Buffer SystemBuffer printed in bold face type is the buffer recommended for optimal activity:A B L M H100% 75-100% 75-100% 75-100% 100%Relative activity in complete PCR mixRelative activity in PCR mix (Taq DNA Polymerase buffer) is 60%. The PCR mix contained ?DNA, primers, 10 mM Tris-HCl (pH 8.3, +20°C), 50 mM KCl, 1.5 mM MgCl2, 200 ?M dNTPs, 2.5 U Taq DNA polymerase. The mix was subjected to 25 amplification cycles. Activity in reaction buffer of Pwo SuperYield DNA Polymerase PCR Mix is 25%. When supplemented with GC-RICH Solution, activity increases to 100%.Incubation temperature+37°CNumber of cleavage sites on different DNAsλ Ad2 SV40 ?X174 M13mp7 M13mp18 pBR322 pBR328 pUC181 5 0 0 0 1 0 0 1PFGE testedXba 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 10 U enzyme per ?g DNA and approximately 4 hour incubation time.Ligation and recutting assayXba I fragments obtained by complete digestion of 1 ?g pUC18 DNA are ligated with 1 U T4 DNA Ligase in a volume of 10 ?l by incubation for 16 hours at +4°C in 66 mM Tris-HCl, 5 mM MgCl2, 5 mM Dithiothreitol, 1 mM ATP, pH 7.5 (at +20°C) resulting in >90% recovery of pUC18 DNA.Subsequent re-cutting with Xba I yields >95% of the typical pattern of pUC18 × Xba I fragments.

Other Notes

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

Specificity

Star ActivityThe sequence specificity of Xba I is relaxed at low ionic strength or by addition of glycerol, ethanol or DMSO to the incubation mixture.Recognition sites: TCTAGATCTAGARestriction site: T↓CTAGAT↓CTAGAHeat inactivation: Xba I can be heat inactivated by incubation at 65 °C for 15 minutes (up to 15 U/µg DNA). Higher concentrations of Xba I cannot be heat inactivated completely under these conditions.

Unit Definition

One unit is the enzyme activity that completely cleaves 1 µg λdam^– DNA in one hour at +37 °C in a total volume of 50 µl (1x) SuRE/Cut Buffer H.