Thermostable FEN1             

Thermostable Flap Endonuclease 1, FEN1, catalyzes the cleavage of 5´ DNA flaps from branched double stranded DNA substrates, creating a 5´ phosphate terminus. FEN1 products can be ligated by DNA ligase to create double stranded DNA. In vivo, FEN1 is an essential component of the Okazaki fragment maturation pathway, and also plays a role in base excision repair. 



Reaction Schematic Thermostable FEN1 recognizes and cleaves 5´ DNA flaps generating a ligatable 5´ phosphate terminus.

Product Source

An E. coli strain, which carries the cloned Thermococcus sp. (strain 9°N^™ FEN1 gene).

Reagents Supplied

The following reagents are supplied with this product:

Applications

• Cleavage of flap DNA structure

Unit Definition

One unit is defined as the amount of enzyme required to cleave 10 pmol of 5´ flap containing oligonucleotide substrate in a total reaction volume of 10 µl for 10 min at 65°C.

Reaction Conditions

1X ThermoPol® Reaction Buffer
Incubate at 65°C

1X ThermoPol® Reaction Buffer:
20 mM Tris-HCl
10 mM (NH₄)₂SO₄
10 mM KCl
2 mM MgSO₄
0.1% Triton® X-100
pH 8.8 @ 25°C

Storage Temperature

-20°C

Storage Conditions

50 mM NaCl
10 mM Tris-HCl
10 mM MgCl₂
1 mM DTT
pH 7.9 @ 25°C

Heat Inactivation

No

Unit Assay Conditions

1X ThermoPol Reaction Buffer containing 0.2 pmol of fluorescently labeled oligonucleotide in a total reaction volume of 10 µl. 

Materials Sold Separately

• ThermoPol® Reaction Buffer Pack

1. Balakrishnon, L. et al. (2013). Annu. Rev. Biochem. 82, 119-138.

2. Greenough, L. et al. (2015). J. Biol. Chem. 20, 12514-12522.

3. Greenough, L. et al. (2015). Nucleic Acids Res. PubMedID: 26365239