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Thermo Scientific™ Klenow Fragment, exo– (5 U/μL)
Description
Klenow Fragment, exo-, is the large fragment of DNA polymerase I . It exhibits 5'→3' polymerase activity, but lacks the 3'→5' and 5'→3' exonuclease activities of DNA Polymerase I. The 3'→5' exonuclease activity of the enzyme is eliminated by mutations in the 3'→5'-exonuclease active sit.
Highlights
- Lacks 3'→5' exonuclease activity
- Incorporates modified nucleotides (e.g., Cy3-, Cy5-, fluorescein-, rhodamine-, aminoallyl-, biotin-labeled nucleotides)
- Active in restriction enzyme, PCR, and RT buffers
Applications
- Random-primed DNA labeling
- Labeling by fill-in 5'-overhangs of dsDNA
- Strand displacement amplification (SDA)
- DNA sequencing by the Sanger method
Note
Klenow Fragment, exo- is not recommended for DNA blunting reactions prior to DNA ligation since it frequently adds one or more extra nucleotides to the 3'-terminus of blunt-end DNA substrates in a non-template directed fashion
Specifications
Specifications
| Concentration | 5 U/μL |
| Polymerase | DNA Polymerase I |
| Compatible Buffer | Restriction Enzyme, PCR, RT Buffer |
| Quantity | 1,500 units |
| Product Type | Klenow Fragment |
Frequently Asked Questions (FAQs)
Thermo Scientific DNA Polymerase I possesses 5′?3′ DNA synthesis activity, 3′?5′ exonuclease (proofreading) activity, and 5′?3′ exonuclease activity. 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. Klenow Fragment, exo-, is also the large fragment of DNA polymerase I. It exhibits 5′?3′ polymerase activity, but lacks the 3′?5′ and 5′?3′ exonuclease activities.
Klenow can be used to "fill in" 5' overhangs of double-stranded DNA fragments using common restriction endonuclease buffers. We recommend REact 2 buffer (1X concentration is 50 mM Tris-HCl pH 8, 50 mM NaCl, 10 mM MgCl2) , but other buffers will also work.
Fill-in Reaction Conditions:
1. Dilute Large Fragment of DNA Polymerase I to 0.5 U/µL with Klenow Dilution Buffer.
2. To a 1.5-mL microcentrifuge tube on ice, add: 10X REact 2 Buffer - 3 µL, 0.5 mM dATP - 1 µL, 0.5 mM dCTP - 1 µL, 0.5 mM dGTP - 1 µL, 0.5 mM dTTP - 1 µL, DNA 0.5-1 µg, Large fragment of DNA Polymerase I - 1 µL, Autoclaved distilled water to 30 µL.
3. Mix gently and centrifuge briefly to bring the contents to the bottom of the tube.
4. Incubate at room temperature for 10-15 minutes or 20 minutes on ice.
5. Terminate fill-in reaction by phenol extraction.
To label the DNA fragment, use 1-2 µL of [alpha-32P]dNTP (400 Ci/mmol, 10 mCi/mL) (24-48 pmoles) instead of the corresponding cold dNTP.
Note: Thermo Fisher Scientific also offers an Exo minus Klenow, which is provided with its own buffers.
For Research Use Only. Not for use in diagnostic procedures.