DNA Polymerase I, Large (Klenow) Fragment
Description:
DNA Polymerase I, Large (Klenow) Fragment is a proteolytic product of E. coli DNA Polymerase I which retains polymerization and 3'- 5' exonuclease activity, but has lost 5'- 3' exonuclease activity. Klenow retains the polymerization fidelity of the holoenzyme without degrading 5' termini.
Source:
The enzyme is produced by directly recombinant the E. coli polA gene, with a deletion of its 5'-3' exonuclease domain. Klenow Fragment is purified with ion exchange and hydrophobic chromatograph. The resulting Klenow fragment has the identical amino and carboxy termini as conventionally prepared Klenow fragment.
Applications:
* DNA sequencing by the Sanger dideoxy method (2)
* Fill-in of 5´ overhangs to form blunt ends (3)
* Removal of 3´ overhangs to form blunt ends (3)
* Second strand cDNA synthesis
* Second strand synthesis in mutagenesis protocols (4).
Enzyme Properties:
* 3´ to 5´ Exonuclease: Yes
* 5´ to 3´ Exonuclease: No
* Strand Displacement: Yes
* Error Rate: 18 x10-6 bases
* Heat Inactivation: 75oC for 20 minutes
* Theoretical MW: 68,000 daltons
* Specific Activity: 20,000 units/mg
* Purity: more than 95% determined by sds-page
Reaction Conditions:
1X KLBuffer
Supplemented with 33 uM dNTPs (not included)
Incubate at 25oC.
1X KLBuffer:
10 mM Tris-HCl
50 mM NaCl
10 mM MgCl2
1 mM Dithiothreitol
pH 7.9 @ 25oC
Unit Definition:
One unit is defined as the same amount of the enzyme from a common used supplier to fill in non-cohesive 5' overhang to allow efficient ligation.
Concentration:
5,000 units/ml and 50,000 units/ml
Storage Conditions:
100 mM KPO4
1 mM Dithiothreitol
50% Glycerol
pH 6.5 @ 25oC
Storage Temperature:
-20oC
Usage notes:
Protocol for blunting ends by 3' overhang removal and 3' recessed end fill-in:
DNA should be dissolved in any 1X KLBuffer supplemented with 33¦ÌM each dNTP. Add 1 unit Klenow per microgram DNA and incubate 15 minutes at 25oC. Stop reaction by adding EDTA to a final concentration of 10mM and heating at 75oC for 20 minutes. CAUTION: Elevated temperatures, excessive amounts of enzyme, failure to supplement with dNTPs or long reaction times may result in recessed ends due to the 3'-5' exonuclease activity of the enzyme.
When DNA Polymerase I, Large (Klenow) Fragment is used to sequence DNA using the dideoxy method of Sanger et al., 1 unit/5ul reaction volume is recommended.
Klenow Fragment is also active in any restriction enzyme reaction buffer and T4 DNA Ligase reaction buffer supplied by other manufacturers when supplemented with dNTPs.
Quality Assurance Statement:
Purified free of contaminating endonucleases and exonucleases. Each lot is functionally tested in "fill-in" reactions. Additionally, each lot is analyzed by SDS polyacrylamide gel electrophoresis for the presence of detectable (less than 1%) contaminants.
Endonuclease Activity:
Incubation of a 50ul reaction containing 50 units of DNA Polymerase I, Large (Klenow) Fragment with 1 ug of pUC118 for 4 hours at 37oC resulted in no obviously pattern changes as determined by agarose gel electrophoresis.
References
1. Jacobsen, H. et al. (1974) Eur. J. Biochem., 45, 623-627.
2. Sanger, F. et al. (1977) Proc. Natl. Acad. Sci. USA, 74, 5463-5467.
3. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, (2nd Ed.), 5.40-5.43.
4. Gubler, U. (1987) S.L. Berger and A.R. Kimmel (Eds.), Methods in Enzymology, 152, pp. 330-335. San Diego: Academic Press.
Description:
DNA Polymerase I, Large (Klenow) Fragment is a proteolytic product of E. coli DNA Polymerase I which retains polymerization and 3'- 5' exonuclease activity, but has lost 5'- 3' exonuclease activity. Klenow retains the polymerization fidelity of the holoenzyme without degrading 5' termini.
Source:
The enzyme is produced by directly recombinant the E. coli polA gene, with a deletion of its 5'-3' exonuclease domain. Klenow Fragment is purified with ion exchange and hydrophobic chromatograph. The resulting Klenow fragment has the identical amino and carboxy termini as conventionally prepared Klenow fragment.
Applications:
* DNA sequencing by the Sanger dideoxy method (2)
* Fill-in of 5´ overhangs to form blunt ends (3)
* Removal of 3´ overhangs to form blunt ends (3)
* Second strand cDNA synthesis
* Second strand synthesis in mutagenesis protocols (4).
Enzyme Properties:
* 3´ to 5´ Exonuclease: Yes
* 5´ to 3´ Exonuclease: No
* Strand Displacement: Yes
* Error Rate: 18 x10-6 bases
* Heat Inactivation: 75oC for 20 minutes
* Theoretical MW: 68,000 daltons
* Specific Activity: 20,000 units/mg
* Purity: more than 95% determined by sds-page
Reaction Conditions:
1X KLBuffer
Supplemented with 33 uM dNTPs (not included)
Incubate at 25oC.
1X KLBuffer:
10 mM Tris-HCl
50 mM NaCl
10 mM MgCl2
1 mM Dithiothreitol
pH 7.9 @ 25oC
Unit Definition:
One unit is defined as the same amount of the enzyme from a common used supplier to fill in non-cohesive 5' overhang to allow efficient ligation.
Concentration:
5,000 units/ml and 50,000 units/ml
Storage Conditions:
100 mM KPO4
1 mM Dithiothreitol
50% Glycerol
pH 6.5 @ 25oC
Storage Temperature:
-20oC
Usage notes:
Protocol for blunting ends by 3' overhang removal and 3' recessed end fill-in:
DNA should be dissolved in any 1X KLBuffer supplemented with 33¦ÌM each dNTP. Add 1 unit Klenow per microgram DNA and incubate 15 minutes at 25oC. Stop reaction by adding EDTA to a final concentration of 10mM and heating at 75oC for 20 minutes. CAUTION: Elevated temperatures, excessive amounts of enzyme, failure to supplement with dNTPs or long reaction times may result in recessed ends due to the 3'-5' exonuclease activity of the enzyme.
When DNA Polymerase I, Large (Klenow) Fragment is used to sequence DNA using the dideoxy method of Sanger et al., 1 unit/5ul reaction volume is recommended.
Klenow Fragment is also active in any restriction enzyme reaction buffer and T4 DNA Ligase reaction buffer supplied by other manufacturers when supplemented with dNTPs.
Quality Assurance Statement:
Purified free of contaminating endonucleases and exonucleases. Each lot is functionally tested in "fill-in" reactions. Additionally, each lot is analyzed by SDS polyacrylamide gel electrophoresis for the presence of detectable (less than 1%) contaminants.
Endonuclease Activity:
Incubation of a 50ul reaction containing 50 units of DNA Polymerase I, Large (Klenow) Fragment with 1 ug of pUC118 for 4 hours at 37oC resulted in no obviously pattern changes as determined by agarose gel electrophoresis.
References
1. Jacobsen, H. et al. (1974) Eur. J. Biochem., 45, 623-627.
2. Sanger, F. et al. (1977) Proc. Natl. Acad. Sci. USA, 74, 5463-5467.
3. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, (2nd Ed.), 5.40-5.43.
4. Gubler, U. (1987) S.L. Berger and A.R. Kimmel (Eds.), Methods in Enzymology, 152, pp. 330-335. San Diego: Academic Press.
