ssDNA/RNA CircLigase

Description

During cyclization, 5' - phosphate group and 3' - OH group are required in the substrate single stranded DNA or RNA. For single stranded substrates larger than 15 nt, ssDNA/RNA CircLigase works efficiently. The enzyme can also catalyze the ligation between DNA or RNA molecules and pre-adenylated DNA junctions. In addition, in the case of excessive ATP, the enzyme can adenosylate DNA/RNA molecules.

Contents

2KU

• 1×CircLigase Buffer: 30 mM Tris-acetate (pH 7.5), 60 mM potassium acetate, 1 mM DTT.

Unit Definition

One unit is defined as the amount of enzyme required to catalyze 1 pmol of a linear 5′-phosphorylated control oligo (55 mer) into circular ssDNA in 1 hour at 60°C.

Note

• The addition of 50 μm ATP can improve the cyclization efficiency, while the addition of ATP with concentration higher than 1 mM can inhibit the ligation.

• For the ligation of single stranded DNA products, the final concentration of 2.5 mm MnCl₂ should be added.

• MnCl₂ is not needed for the ligation of single stranded RNA products.

• For the substrates with secondary or complex structure, 0.4 ~ 0.8 m Betain can be added to improve the efficiency.

• The enzyme has selectivity for substrate base. 5' terminal ligation efficiency: G > A > > T(U) > > C, 3' terminal ligation efficiency: T(U) > A > G. When the 3' terminal base is C, it cannot be used for ligation, as there will be almost no ligation products.

Storage

Minimum shelf life is 3 year at -20°C.

Instruction: Protocol

Only for research and not intended for treatment of humans or animals