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  PCR & DNA modifying enzymes

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)

60.00144.00
  • 5u/μl
  • 3×400 U
  • 400 U

 

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 25o
  • 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 -20o

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. 

 

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