Applications

• Ligation amplification (Ligase Chain Reaction, LCR)^1-7
• Repeat Expansion Detection (RED)⁷
• Simultaneous mutagenesis of multiple sites^8,9
• Other ligation-based detection methods

Ampligase® Thermostable DNA Ligase catalyzes NAD-dependent ligation of adjacent 3´-hydroxylated and 5´-phosphorylated termini in duplex DNA structures that are stable at high temperatures. Derived from a thermophilic bacterium, the enzyme is stable and active at much higher temperatures than conventional DNA ligases. Its half-life is 48 hours at 65°C and greater than 1 hour at 95°C. Ampligase DNA Ligase has been shown to be active for at least 500 thermal cycles (94°C/80°C) or 16 hours of cycling.¹⁰ This exceptional thermostability permits extremely high hybridization stringency and ligation specificity. Ampligase DNA Ligase has no detectable activity in ligating blunt-ended DNA and has no activity on RNA or RNA:DNA hybrids.

Unit Definition: One unit of Ampligase DNA Ligase catalyzes the ligation of 50% of the cos sites in 1 µg of lambda DNA in 1 minute at 45°C in 1X Ampligase Reaction Buffer.

Note: One unit of Ampligase DNA Ligase is equivalent to at least 15 of the \"cohesive end units\" or \"nick ligation units\" defined elsewhere.⁴

Storage Buffer: 50% glycerol containing 50 mM Tris-HCl (pH 7.5), 0.1 M NaCl, 0.1 mM EDTA, 1 mM DTT, and 0.1% Triton® X-100.

Ampligase 10X Reaction Buffer: 200 mM Tris-HCl (pH 8.3), 250 mM KCl, 100 mM MgCl₂, 5 mM NAD, and 0.1% Triton® X-100.

Quality Control: Ampligase Thermostable DNA Ligase is assayed for the absence of blunt-end ligation activity by ligating Sma I-digested bacteriophage lambda DNA at 45°C; ligation occurs only at cos sites as determined by gel electrophoresis. Ampligase DNA Ligase is free of detectable exo- and endonuclease and RNase activities.

References