Product Specification
| Synonyms | Klenow Fragment、DNA Polymerase I Large (Klenow) Fragment |
| Amino Acid Sequence | / |
| Expression System | E.coli |
| Molecular Weight | 70kDa (Reducing) |
| Purity | ＞95% by SDS-PAGE and HPLC |
| Conjugation | Unconjugated |
| Tag | His Tag |
| Physical Appearance | Liquid |
| Storage Buffer | 25 mM Tris-HCl, 1 mM DTT, 0.1 mM EDTA, 50% Glycerol, pH 7.4 @ 25°C |
| Reconstitution | / |
| Stability & Storage | Store at -25 ~ -15℃ for 2 years |
| Reference | [1] Zhao, Guojie , et al. "Realizing directional cloning using sticky ends produced by 3′-5′ exonuclease of Klenow fragment." Journal of Biosciences 38.5(2013):857-866.
[2] Olsen, Tivoli J. , et al. "Electronic Measurements of Single-Molecule Processing by DNA Polymerase I (Klenow Fragment)." Journal of the American Chemical Society 135.21(2013):7855-7860.
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Background
The Klenow Fragment, is a large fragment of E.coli. DNA polymerase I. It retains the 3'→5' exonuclease activity of DNA polymerase I, but lacks the 5'→3' exonuclease activity of the intact DNA polymerase I. The 3'→5' exonuclease activity of Klenow Fragment ensures accurate proofreading when synthesizing DNA. It is used to fill in the 5'overhang ends of double-stranded DNA; and double-stranded DNA 3'overhang flattening (also called trimming). It can also be used for the synthesis of the second strand of cDNA or the synthesis of the second strand of site-specific mutation reaction.
Components
Storage Solution: 5 U/ul Klenow Fragment、25 mM Tris-HCl, 1 mM DTT, 0.1 mM EDTA, 50% Glycerol, pH 7.4 @ 25°C
10*Reaction Buffer: 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 1 mM DTT
(pH 7.9 @ 25°C)
Protocol
1. DNA should be dissolved in 1*Reaction Buffer or T4 DNA Ligase Reaction buffer and supplemented with 33 μM each dNTP.
2. Add 1 unit of DNA Polymerase I Large (Klenow) Fragment per microgram DNA.
3. Incubate for 15 minutes at 25°C.
4. Stop reaction by adding EDTA to a final concentration of 10 mM and heating for 20 minutes at 75°C.
Guidelines
1. Due to the 3´→5´ exonuclease activity of the enzyme, increasing the reaction temperature, adding too much enzyme, not adding dNTP or too long reaction time will lead to the formation of the dented end.
2. Please avoid repeated freeze-thaw cycles
Unit Definition
One unit is defined as the amount of enzyme required to incorporate 10 nmol of dNTP into acid insoluble substances at 37 ° C for 30 minutes.
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