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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
Related Categories
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.
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.
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.
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
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.
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 Code
12 - Non Combustible Liquids
WGK
WGK 1
Flash Point(F)
does not flash
Flash Point(C)
does not flash
Certificates of Analysis (COA)
Search for Certificates of Analysis (COA) by entering the products Lot/Batch Number. Lot and Batch Numbers can be found on a product’s label following the words ‘Lot’ or ‘Batch’.
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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.
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Restriction endonucleases in prokaryotes function primarily to protect against foreign genetic material, notably bacteriophage DNA.
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