Klenow Fragment

For general laboratory use.

Definição de unidade: One unit is defined as the amount of enzyme required to convert 10 nmoles of dNTPs to an acid insoluble form in 30 minutes at 37 °C.

Envio: shipped on gel packs

Condições de armazenamento: store at -20 °C
avoid freeze/thaw cycles

Validade: 12 months

Forma: liquid (Supplied in 100 mM KPO4 pH 6.5, 1 mM DTT and 50 % [v/v] glycerol)

Concentração: 5 units/μl

Formulários:

• Fill-in of 5' overhangs to form blunt ends
• Removal of 3' overhangs to form blunt ends

Descrição:
Klenow Fragment is the large fragment of DNA Polymerase I that retains its 5'→3' polymerase, 3'→5' exonuclease and strand displacement activities. The enzyme lacks the 5'→3' exonuclease activity of intact DNA polymerase I. Klenow retains the polymerization fidelity of the holoenzyme without degrading 5' termini.

Reaction conditions:Dissolve 0.1 - 4 μg of digested DNA in 1x Reaction Buffer supplemented with 40 μM each dNTPAdd 1 unit Klenow Fragment per μg DNA
Incubate for 15 min. at 25 °C

Stop reaction by alternativlyadd EDTA to 10 mM final concentrationHeat inactivation: 20 min. at 75 °C
10x Reaction Buffer:
500 mM Tris-HCl pH 7.6 at 25 °C, 50 mM MgCl2 and 10 mM DTT.

Note:
Excessive amounts of enzyme or longer reaction times may result in recessed ends due to the 3'→5' exonuclease activity of the enzyme.

Quality Control:
The enzyme is greater than 98 % pure as indicated by SDS-polyacrylamide gel electrophoresis and contains no detected endonuclease activity. Incubation of 10 units of Klenow with supercoiled plasmid DNA produced no nicked molecules after 20 hours at 37 °C as determined by agarose gel electrophoresis analysis.