The experiment was designed to synthesize two complementary DNA Oligos. Small DNA fragments with 5 'sticky ends were synthesized by annealing, and the sticky ends were completed by Klenow fragment to form flat ends. 20% Native PAGE glue (which can distinguish the size of 0.1 ~ 100bp) was used for electrophoresis, and the enzyme activity was verified. As can be seen from the result, the dsDNA fragment at the sticky end is mended by Klenow fragment, which has a larger molecular weight, and the electrophoretic band position is above the DNA fragment with the sticky end. The results show that UA070056-Klenow fragment has the same effect as a competing product N.
Lane 1 Negative Control
Lane 2 competing product N 0.44μg
Lane 3 competing product N 0.22μg
Lane 4 UA070056-Klenow fragment 0.44μg
Lane 5 UA070056-Klenow fragment 0.22μg
Lane 6 UA070056-Klenow fragment 0.42μg
Lane 7 UA070056-Klenow fragment 0.21μg
DNA Polymerase I Large (Klenow) Fragment
DNA Polymerase I Large (Klenow) Fragment
Product Details
Product Details
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. |
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
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Bioactivity
SDS-PAGE
1μg (R: reducing condition, N: non-reducing condition).
RP-HPLC
