Xba I

Кат. номер

10674265001

11047663001

10674257001

10674273001

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 ends
Xba I ends are compatible with fragments generated by Avr I, Nhe I, and Spe I.
Isoschizomers
The enzyme is not known to have isoschizomers.
Methylation sensitivity
Xba 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 System
Buffer printed in bold face type is the buffer recommended for optimal activity:
A B L M H
100% 75-100% 75-100% 75-100% 100%
Relative activity in complete PCR mix
Relative 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°C
Number of cleavage sites on different DNAs
Ad2 SV40 ?X174 M13mp7 M13mp18 pBR322 pBR328 pUC18
1 5 0 0 0 1 0 0 1
PFGE tested
Xba 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 assay
Xba 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 x Xba I fragments.
Specificity
Star Activity
The 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: TCTAGA
TCTAGA
Restriction site: TCTAGA
TCTAGA
Heat 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.
The restriction enzyme Xba I has been used for the digestion of genomic DNA.
DNA Profile
Number of cleavage sites on different DNAs

• : 1
• X174: 0
• Ad2: 5
• M13mp7: 0
• pBR322: 0
• pBR328: 0
• pUC18: 1
• SV40: 0

Unit Definition
One unit is the enzyme activity that completely cleaves 1 µg dam– DNA in one hour at +37 °C in a total volume of 50 µl (1x) SuRE/Cut Buffer H.
Analysis Note
Absence of nonspecific endonuclease activities
1µg DNA 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 activity
Approximately 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.
SuRE/Cut Buffer System
The buffer in bold is recommended for optimal activity

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

Activity in PCR buffer: 60%
Relative 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 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.
For life science research only. Not for use in diagnostic procedures.

Quality Level	100,
form	solution
packaging	pkg of 1,000 U (10674257001 [10 U/µl]), pkg of 20,000 U (10674273001 [10 U/µl]), pkg of 20,000 U (11047663001 [40 U/µl]), pkg of 5,000 U (10674265001 [10 U/µl])
Торговая марка	Roche
parameter	37 °C optimum reaction temp.
shipped in	dry ice
storage temp.	20°C

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NONH for all modes of transport