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)
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Product Details
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.
Applications:
- Fill-in of 5′ overhangs to form blunt ends
- Removal of 3′ overhangs to form blunt ends
Reaction Guidelines
- 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.
Storage conditions:
0.1 M KPO4 (pH 6.5)
1 mM DTT
50% glycerol
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.
