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DNA Polymerase I, Large (Klenow) Fragment |
 | | | | DNA Polymerase I, Large (Klenow) Fragment | | | |
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Prices are in US dollars and valid only for US orders.
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- Isolated from a recombinant source
- Specific Activity: 20,000 units/mg
- Dideoxy sequencing
- Generates probes using random primers
- Creates blunt ends
- Supplied with 10X Reaction Buffer
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:
A genetic fusion of the E. coli polA gene, that has had its 5'→3' exonuclease domain genetically replaced by maltose binding protein (MBP). Klenow Fragment is cleaved from the fusion and purified away from MBP. 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)
- Second strand cDNA synthesis
- Second strand synthesis in mutagenesis protocols (4).
- Removal of 3´ overhangs to form blunt ends (3)
Reagents Supplied:
NEBuffer 2 (10X)
Enzyme Properties
Polymerase Properties | Thermophilic Polymerase Characteristics
3´ to 5´ Exonuclease: Yes
5´ to 3´ Exonuclease: No
Strand Displacement: Yes
Error Rate: 18
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
25°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 form in 30 minutes at 37°C.
Unit assay Conditions:1X NEBuffer 2, 33 μM dNTPs including [3H]-dTTP and 70 μg/ml denatured herring sperm DNA.
DNA Sequencing:
When this preparation is used to sequence DNA using the dideoxy method of Sanger et al., 1 unit/5 μl reaction volume is recommended.
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:- Protocol for blunting ends by 3' overhang removal and 3' recessed end fill-in:
DNA should be dissolved in any 1X restriction enzyme NEBuffer or 1X EcoPol Reaction Buffer supplemented with 33μM each dNTP. Add 1 unit Klenow per microgram DNA and incubate 15 minutes at 25°C. Stop reaction by adding EDTA to a final concentration of 10mM and heating at 75°C 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/5 μl reaction volume is recommended.
- Klenow Fragment is also active in any restriction enzyme reaction buffer and T4 DNA Ligase reaction buffer when supplemented with dNTPs.
FAQs
- Can DNA Polymerase I, Large (Klenow) Fragment be used in other NEBuffers?
- Can DNA Polymerase I, Large (Klenow) Fragment be used to blunt DNA?
- Can DNA Polymerase I, Large (Klenow) Fragment be used to fill in 3' overhangs?
- Can DNA Polymerase I, Large (Klenow) Fragment be used to chew back 5' overhangs?
- Are NEB DNA Polymerases supplied with dNTPs?
- Can DNA Polymerase I, Large (Klenow) Fragment be heat inactivated?
- Are the nucleotides needed to remove a 3' overhang with DNA Polymerase I, Large (Klenow) Fragment?
- What are the main causes for blunting reaction failure using DNA Polymerase I, Large (Klenow) Fragment?
- Can DNA Polymerase I, Large (Klenow) Fragment be used in labeling reactions and partial fill in reactions?
- Is DNA Polymerase I, Large (Klenow) Fragment 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 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%) uncleaved DNA Polymerase I.
Endonuclease Activity:
Incubation of a 50 μl reaction containing 50 units of DNA Polymerase I, Large (Klenow) Fragment 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.
References
- Jacobsen, H. et al. (1974) Eur. J. Biochem., 45, 623-627.
- Sanger, F. et al. (1977) Proc. Natl. Acad. Sci. USA, 74, 5463-5467.
- Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, (2nd Ed.), 5.40-5.43.
- 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
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