DNA Polymerase I Large Fragment (Klenow Fragment)
Klenow Fragment is purified from an E. coli strain carrying a DNA Polymerase I large fragment overproducing plasmid.
Reagents supplied: 10x Klenow buffer
Unit definition: 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 37oC.
(SKUs: PD004S, PD004L)
The Klenow Fragment lacks the 5′→3′exonuclease activity of intact DNA Polymerase I but retains the 5′→3′polymerase, the 3′→5′exonuclease and the strand displacement activities.
- Fill-in of 5′ overhangs to form blunt ends
- Removal of 3′ overhangs to form blunt ends
- Reaction conditions: Dissolve 0.1-4 μg of digested DNA in 1x Klenow Reaction Buffer supplemented with 40μM each dNTP. Add 1 unit Klenow per μg DNA.
Incubate for 15 min at 25oC.
|Components||20μl assay||Final Concentration/Quantity|
|10x Klenow buffer||2μl||1x|
|dNTPs mix/2mM each||0.4μl||40μM each dNTP|
|Digested DNA||Variable||0.1-4 μg|
|Klenow fragment (5u/μl)||Variable||1u per μg DNA|
|Sterile ultrapure water||Up to 20 μl|
|Incubate for 15 min at 25oC|
- Stop reaction: Add EDTA to 10 mM final concentration and heat at 75oC for 10 min.
10x Klenow buffer: 500 mM Tris-HCl (pH 7.6 @ 25oC), 50 mM MgCl2 and 10 mM DTT
Klenow fragment is also 50% active in all five standard EnzyQuest buffers when supplemented with dNTPs.
0.1 M KPO4 (pH 6.5)
1 mM DTT
Store at -20oC
Heat inactivation: 75oC for 20 minutes
Quality controls: Tested extensively for the absence of endo- and exodeoxyribonucleases. More details concerning quality controls could be found in Certificate of analysis.
Note: Excessive amounts of enzyme or longer reaction times may result in recessed ends due to the 3′→5′exonuclease activity of the enzyme.