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XBAI-RO

Roche

Xba I

from recombinant E.Coli

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biological source

Escherichia coli

Quality Level

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])

manufacturer/tradename

Roche

parameter

37 °C optimum reaction temp.

technique(s)

DNA sequencing: suitable

storage temp.

−20°C

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 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
λ 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 × 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: T↓CTAGA
T↓CTAGA
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.

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

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.

Analysis Note

Absence of nonspecific endonuclease activities
1μg 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 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 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.

Other Notes

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

Kit Components Only

Product No.
Description

  • Enzyme Solution

  • SuRE/Cut Buffer H 10x concentrated

Storage Class

12 - Non Combustible Liquids

wgk_germany

WGK 1

flash_point_f

does not flash

flash_point_c

does not flash


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Articles

The term “Restriction enzyme” originated from the studies of Enterobacteria phage λ (lambda phage) in the laboratories of Werner Arber and Matthew Meselson.

Related Content

Restriction endonucleases in prokaryotes function primarily to protect against foreign genetic material, notably bacteriophage DNA.

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