Apa I

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.

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.

Apa I recognizes the sequence GGG*CC?*C and generates fragments with 3′-cohesive termini.

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.

Number of cleavage sites on different DNAs

• λ: 1
• φX174: 0
• Ad2: 12
• M13mp7: 0
• pBR322: 0
• pBR328: 0
• pUC18: 0
• SV40: 1

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.

SuRE/Cut Buffer System
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.

仅用于生命科学研究。不可用于诊断。