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FAQs for Klenow Fragment (3´→5´ exo–) FAQs for Polymerases Technical Reference for Polymerases Enzyme Finder NEBcutter NEBuffer Chart Double Digest Finder Isoschizomers DNA Sequences and Maps REBASE NEBuffer 2

Home > Products > Polymerases > Mesophilic DNA Polymerases > Klenow Fragment (3´→5´ exo–) Klenow Fragment (3´→5´ exo–) Catalog # Size Concentration Price Qty   M0212L 1,000 units 5,000 units/ml $224.00 M0212M 1,000 units 50,000 units/ml $224.00 M0212S 200 units 5,000 units/ml $56.00 Prices are in US dollars and valid only for US orders. Download: Technical Bulletin|MSDS PDF Isolated from a recombinant source Generates probes using random primers Specific Activity: 20,000 units/mg Dideoxy sequencing Supplied with 10X Reaction Buffer Moderate strand displacement activity Description: Klenow Fragment (3´→ 5´ exo- ) is an N-terminal truncation of DNA Polymerase I which retains polymerase activity, but has lost the 5´→ 3´ exonuclease activity and has a mutation (D355A, E357A) which abolishes the 3´→ 5´ exonuclease activity (1). Source: An E. coli strain containing a plasmid with a fragment of the E.coli polA (D355A, E357A) gene starting at codon 324. Applications: Random priming labeling DNA sequencing by the Sanger dideoxy method (2) Second strand cDNA synthesis Second strand synthesis in mutagenesis protocols (3). Reagents Supplied: NEBuffer 2 Enzyme Properties Polymerase Properties | Thermophilic Polymerase Characteristics 3´ to 5´ Exonuclease: No 5´ to 3´ Exonuclease: No Strand Displacement: Yes Error Rate: 100 x10-6 bases Heat Inactivation: 75°C for 20 minutes Molecular Weight: Theoretical: 68,000 daltons Specific Activity: 20,000 units/mg Reaction & Storage Conditions Reaction Conditions: 1X NEBuffer 2 Supplemented with 33 μM dNTPs (not included) Incubate at 37°C. 1X NEBuffer 2: 10 mM Tris-HCl 50 mM NaCl 10 mM MgCl2 1 mM Dithiothreitol pH 7.9 @ 25°C Unit Definition: One unit is defined as the amount of enzyme required to convert 10 nmol of dNTPs to an acid-insoluble material in 30 minutes at 37°C. Concentration: 5,000 units/ml and 50,000 units/ml Storage Conditions: 25 mM Tris-HCl 1 mM Dithiothreitol 0.1 mM EDTA 50% Glycerol pH 7.4 @ 25°C Storage Temperature: -20°C Notes Usage notes: Klenow Fragment (3´→ 5´ exo- ) is not suitable for generating blunt ends because it lacks the 3´→ 5´ exonuclease necessary to remove non-templated 3´ additions. Klenow Fragment (3´→5´ exo-) is also active in all NEB restriction enzyme reaction buffers when supplemented with dNTPs. Application notes: When Klenow Fragment (3´→ 5´ exo- ) is used to sequence DNA using the dideoxy method of Sanger et al. 1 unit/5 µl reaction volume is recommended. FAQs Can Klenow Fragment (3'→5' exo-) be used in other NEBuffers? Can Klenow Fragment (3'→5' exo-) be used to blunt DNA? Can Klenow Fragment (3'→5' exo-) be used to fill in 3' overhangs? Can Klenow Fragment (3'→5' exo-) be used to remove 5' overhangs? Can Klenow Fragment (3'→5' exo-) be heat inactivated? Can Klenow Fragment (3'→5' exo-) be used in labeling reactions and partial fill in reactions? Are NEB DNA Polymerases supplied with dNTPs? Are there other methods for making probes? What is the enzyme of choice for chewing back 3' overhangs and filling in 5' overhangs (3' recessed ends)? Quality Control for Current Lot Quality control values for a specific lot can be found on the datacard which accompanies each product. Quality Assurance Statement: Purified free of contaminating endonuclease and exonuclease. Exonuclease Activity: Incubation of a 50 μl reaction containing 200 units of Klenow Fragment (3´→5´ exo–) with 1 μg of a mixture of single and double-stranded [3H] E. coli DNA (205 cpm/μg) for 4 hours at 37ºC released < 0.1% of the total radioactivity. Endonuclease Activity: Incubation of a 50 μl reaction containing 50 units of Klenow Fragment (3´→5´ exo–) with 1 μg of ΦX174 RF I DNA for 4 hours at 37ºC resulted in < 10% conversion to RFII as determined by agarose gel electrophoresis. 3' → 5' Exonuclease Activity: Incubation of 50 units of Klenow (exo-) in 20 µl of a 10 nM solution of a fluorescent 5’-FAM labeled oligonucleotide for 30 minutes at 37°C yields no detectable 3’-->5’ degradation as determined by high resolution denaturing PAGE. References Derbyshire, V. et al. (1988) Science, 240, 199-201. Sanger, F. et al. (1977) Proc. Natl. Acad. Sci. USA, 74, 5463-5467. Gubler, U. (1987) S.L. Berger and A.R. Kimmel (Eds.), Methods in Enzymology, 152, pp. 330-335. San Diego: Academic Press. Reagents Sold Separately NEBuffer 2