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Molecular Labeling & Detection

Klenow Fragment

Product information

Features

• Incorporates modified nucleotides (e.g., Cy3-, Cy5-, aminoallyl-, biotin-, digoxigenin- and fluorescently-labeled nucleotides).
• Active in restriction enzyme, PCR, RT and T4 DNA Ligase buffers.

Applications

• DNA blunting by fill-in 5'-overhangs (1).
• Random-primed DNA labeling (2-4).
• Labeling by fill-in 5'-overhangs of dsDNA.
• DNA sequencing by the Sanger method (5).
• Site-specific mutagenesis of DNA with synthetic oligonucleotides (6).
• Second strand synthesis of cDNA (7).

Description

Klenow Fragment is the Large Fragment of DNA Polymerase I. It exhibits 5'=>3' polymerase activity and 3'=>5' exonuclease (proofreading) activity, but lacks 5'=>3' exonuclease activity of DNA polymerase I.

Source

E.coli cells with a cloned fragment of the polA gene.

Molecular Weight

68 kDa monomer.

Definition of Activity Unit

One unit of the enzyme catalyzes the incorporation of 10 nmol of deoxyribonucleotides into a polynucleotide fraction (adsorbed on DE-81) in 30 min at 37°C, using poly(dA-dT)·poly(dA-dT) as a template·primer.

Enzyme activity is assayed in the following mixture: 67 mM potassium phosphate (pH 7.4), 6.7 mM MgCl₂, 1 mM 2-mercaptoethanol, 0.033 mM dATP, 0.033 mM dTTP, 0.4 MBq/ml [³H]-dTTP and 62.5 µg/ml poly(dA-dT)·poly(dA-dT).

Storage Buffer

The enzyme is supplied in:
25 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 1 mM DTT and 50% (v/v) glycerol.

10X Reaction Buffer

500 mM Tris-HCl (pH 8.0 at 25°C), 50 mM MgCl₂, 10 mM DTT.

Quality Control

The absence of endodeoxyribonucleases confirmed by appropriate quality test. Functionally tested for fill in of 5'-overhanging DNA termini and for random primed DNA labeling.

Inhibition and Inactivation

• Inhibitors: metal chelators, PP_i, P_i (at high concentrations) (8).
• Inactivated by heating at 75°C for 10 min or by addition of EDTA.

Patents, Licenses, Trademarks

Protocols & recommendations

RECOMMENDATIONS FOR USE

ADDITIONAL PROTOCOLS

Fill-in of 5'-overhangs with Klenow Fragment

1. Prepare the following reaction mixture:
   

   Linear DNA	10-15 µl (0.1-4 µg)
   10X reaction buffer for Klenow Fragment	2 µl
   dNTP Mix, 2 mM each	0.5 µl (0.05 mM final concentration)
   Klenow Fragment	0.1-0.2 µl (1-5 u)
   Water, nuclease-free	to 20 µl
   Total volume	20 µl

2. Mix thoroughly, spin briefly and incubate at 37°C for 10 min.
3. Stop the reaction by heating at 75°C for 10 min.

DNA 3'-end Labeling by Fill-in of 5'-overhangs with Klenow Fragment* or Bsm DNA Polymerase, Large Fragment

1. Prepare the following reaction mixture:

   Linear DNA (aqueous solution)	0.1-4 µg
   10X reaction buffer for Klenow Fragment or 10X Bsm buffer	2 µl
   [alpha-32P]-dNTP, ~15-30 TBq/mmol (400-800 Ci/mmol) or [alpha-32P]-dNTP, ~110 TBq/mmol (3000 Ci/mmol)	0.74 MBq (20 µCi) 2.96 MBq (80 µCi)
   3 dNTP Mix, 2 mM each (without a labeled dNTP)	2.5 µl (0.25 mM final concentration)
   Klenow Fragment or Klenow Fragment, exo- or Bsm DNA Polymerase, Large Fragment	0.1 µl (1 u) 0.2 µl (1 u) 0.125 µl (1 u)
   Water, nuclease-free	to 20 µl
   Total volume	20 µl

2. Incubate at 37°C for 15 min.
3. Stop the reaction by heating at 75°C for 10 min.

Note

* This protocol is suitable labeling of the following Fermentas DNA markers, composed of DNA fragments with 5'-overhangs:
Lambda DNA EcoRI Marker, #SM028
Lambda DNA HindIII Marker, #SM0101
Lambda DNA EcoRI/HindIII Marker, #SM0191
Lambda DNA Eco91I Marker, #SM0111
phiX174 DNA HinfI Marker, #SM0261

• The modified version of this protocol can be used for non-radioactive labeling of DNA markers. Substitute a part of dTTP nucleotide with a modified nucleotide (e.g. Biotin-11-dUTP or Fluorescein-12-dUTP) at a molar ratio of 1:2.
• For estimation of pmol of DNA ends, see REviewer™.

References

1. Ausubel, F.M., et al., Current Protocols in Molecular Biology, vol. 1, John Wiley & Sons, Inc., Brooklyn, New York, 3.5.7-3.5.10, 1994-2005.
2. Feinberg, A.P., Vogelstein, B., A technique for radiolabeling DNA restriction endonucleases fragments to high specific activity, Anal. Biochem., 132, 6-13, 1983.
3. Feinberg, A.P., Vogelstein, B., Addendum to: A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity, Anal. Biochem., 137, 266-267, 1984.
4. Yu, H., et al., Cyanine dye dUTP analogs for enzymatic labeling of DNA probes, Nucleic Acids Res., 22, 3226-3232, 1994.
5. Sanger, F., et al., DNA sequencing with chain-terminating inhibitors, Proc. Natl. Acad. Sci. USA, 74, 5463-5467, 1977.
6. Wallace, R.B., et al., Directed deletion of a yeast transfer RNA intervening sequence, Science, 209, 1396-1400, 1980.
7. Rougeon, F., et al., Insertion of rabbit beta-globin gene sequence into an E.coli plasmid, Nucleic Acids Res., 2, 2365-2378, 1975.
8. Eun, H-M., Enzymology Primer for Recombinant DNA Technology, Academic Press, Inc., 1996.

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