APAI-RO
Roche
Apa I
from Acetobacter pasteurianus
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About This Item
Code UNSPSC :
12352204
Produits recommandés
Forme
solution
Conditionnement
pkg of 20,000 U (10703753001 [40 U/μl])
pkg of 5,000 U (10899208001 [10 U/μl])
Fabricant/nom de marque
Roche
Paramètres
30 °C optimum reaction temp.
Conditions d'expédition
dry ice
Température de stockage
−20°C
Description générale
Compatible ends
Apa I ends are not compatible with those generated by any other known restriction enzymes.
Isoschizomers
Apa I is an isoschizomer to Bsp 120 I and Psp OMI.
Methylation sensitivity
Apa I is inhibited by 5′-methylcytosine at the sites indicated (*) in the recognition sequence.
Activity in SuRE/Cut Buffer System
Buffer printed in bold face type is the buffer recommended for optimal activity:
ABLMH
100%10-25%50-75%50-75%0-10%
Relative activity in complete PCR mix
Relative activity in PCR mix (Taq DNA Polymerase buffer) is 100%. The PCR mix contained target 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 10%. When supplemented with GC-RICH Solution activity is increased to >100%.
Incubation temperature
+30°C
Unit definition
One unit is the enzyme activity that completely cleaves 1 μg ? × Hind III fragments in 1 hour at +30°C in a total volume of 25 μl SuRE/Cut Buffer A.
Heat inactivation
The enzyme can be heat inactivated by incubating it for 15 minutes at +65°C.
Number of cleavage sites on different DNAs
λAd2SV40φ X174M13mp7M13mp18pBR322pBR328pUC18
112 1000000
Ligation and recutting assay
Apa I fragments obtained by complete digestion of 1 μg λ × Hind III fragments are ligated with 1 U T4 DNA Ligase in a volume of 10 μl 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 >95% recovery of 1 μg λ × Hind III fragments.
Subsequent re-cutting with Apa I yields >95% of the typical pattern of λ × Hind III× Apa I fragments.
Apa I ends are not compatible with those generated by any other known restriction enzymes.
Isoschizomers
Apa I is an isoschizomer to Bsp 120 I and Psp OMI.
Methylation sensitivity
Apa I is inhibited by 5′-methylcytosine at the sites indicated (*) in the recognition sequence.
Activity in SuRE/Cut Buffer System
Buffer printed in bold face type is the buffer recommended for optimal activity:
ABLMH
100%10-25%50-75%50-75%0-10%
Relative activity in complete PCR mix
Relative activity in PCR mix (Taq DNA Polymerase buffer) is 100%. The PCR mix contained target 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 10%. When supplemented with GC-RICH Solution activity is increased to >100%.
Incubation temperature
+30°C
Unit definition
One unit is the enzyme activity that completely cleaves 1 μg ? × Hind III fragments in 1 hour at +30°C in a total volume of 25 μl SuRE/Cut Buffer A.
Heat inactivation
The enzyme can be heat inactivated by incubating it for 15 minutes at +65°C.
Number of cleavage sites on different DNAs
λAd2SV40φ X174M13mp7M13mp18pBR322pBR328pUC18
112 1000000
Ligation and recutting assay
Apa I fragments obtained by complete digestion of 1 μg λ × Hind III fragments are ligated with 1 U T4 DNA Ligase in a volume of 10 μl 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 >95% recovery of 1 μg λ × Hind III fragments.
Subsequent re-cutting with Apa I yields >95% of the typical pattern of λ × Hind III× Apa I fragments.
Quality
Absence of nonspecific endonucleases
1 μg λ × Hind III fragments is incubated for 16 hours in 50 μl SuRE/Cut Buffer A with excess Apa 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 3 μl Apa 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.
Absence of nonspecific endonucleases
1 μg λ × Hind III fragments is incubated for 16 hours in 50 μl SuRE/Cut Buffer A with excess Apa 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 3 μl Apa 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.
Apa I recognizes the sequence GGG*CC?*C and generates fragments with 3′-cohesive termini.
Spécificité
Recognition sites: GGG*CC*C
GGG*CC*C
Restriction site: GGG*CC↓*C
GGG*CC↓*C
Heat inactivation: Apa I can be heat inactivated by incubation at 65 °C for 15 minutes.
GGG*CC*C
Restriction site: GGG*CC↓*C
GGG*CC↓*C
Heat inactivation: Apa I can be heat inactivated by incubation at 65 °C for 15 minutes.
Profil d'ADN
Number of cleavage sites on different DNAs
- λ: 1
- φX174: 0
- Ad2: 12
- M13mp7: 0
- pBR322: 0
- pBR328: 0
- pUC18: 0
- SV40: 1
Définition de l'unité
One unit is the enzyme activity that completely cleaves 1 μg HindIII fragments of λDNA in one hour at +30 °C in a total volume of 25 μl (1x) SuRE/Cut Buffer A.
Remarque sur l'analyse
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: 10-25%
- H: 0-10%
- L: 50-75%
- M: 50-75%
Activity in PCR buffer: 100%
Relative activity in PCR mix (Taq DNA Polymerase buffer) is 100%. The PCR mix contained target 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 10%. When supplemented with GC-RICH Solution activity is increased to > 100%. Pwo SuperYield DNA Polymerase PCR Mix is not available in U.S.
Relative activity in PCR mix (Taq DNA Polymerase buffer) is 100%. The PCR mix contained target 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 10%. When supplemented with GC-RICH Solution activity is increased to > 100%. Pwo SuperYield DNA Polymerase PCR Mix is not available in U.S.
Autres remarques
For life science research only. Not for use in diagnostic procedures.
Composants de kit seuls
Réf. du produit
Description
- Enzyme Solution
- SuRE/Cut Buffer A 10x concentrated
Certificats d'analyse (COA)
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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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