Pst I

Absence of nonspecific endonuclease activities
1 μg λDNA is incubated for 16 hours in 50 μl SuRE/Cut Buffer H with an excess of Pst 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 Pst 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: 25-50%
• B: 25-50%
• H: 100%
• L: 10-25%
• M: 25-50%

Activity in PCR buffer: 90%

Relative activity in PCR mix (Taq DNA Polymerase buffer) is less than 90%. 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 35%. When supplemented with GC-RICH Solution, activity drops to 15%. Pwo SuperYield DNA Polymerase PCR Mix is not available in U.S.

Number of cleavage sites on different DNAs

• λ: 28
• φX174: 1
• Ad2: 30
• M13mp7: 1
• pBR322: 1
• pBR328: 1
• pUC18: 1
• SV40: 2

The recognition specificity of Pst I is altered by addition of increasing amounts of hydrophobic reagents and glycerol to the incubation mixture.
Recognition sites: CTGCAG
CTGCAG
Restriction site: CTGCA↓G
CTGCA↓G
Heat inactivation: No Inactivation of Pst I by incubation at 65 °C for 15 minutes.

Compatible ends
Pst I generates ends that are compatible with fragments generated by Asp I, AspH I, and Nsi I.

Isoschizomers
The enzyme is not known to have isoschizomers.

Methylation sensitivity
Pst I is inhibited by the presence of 5-methylcytosine and 6-methyladenine at the sites indicated (*) on the recognition sequence.

Incubation temperature
+37°C

Star activity
The recognition specificity of Pst I is altered when increasing amounts of hydrophobic reagents and glycerol are added to the incubation mixture.

Ligation and recutting assay
Pst I fragments obtained by complete digestion of 1 μg λDNA 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 λDNA fragments.
Subsequent re-cutting with Pst I yields >95% of the typical pattern of λDNA × Pst I fragments.

Pst I recognizes the sequence *CTGC°A↓G and generates fragments with 3′-cohesive termini.
Contents:

• Pst I
• SuRE/Cut Buffer H (10x)

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

One unit is the enzyme activity that completely cleaves 1 μg λDNA in one hour at +37 °C in a total volume of 25 μl (1x) SuRE/Cut buffer H.