Klenow Fragment, Exo-

Item Number

K747451

• Specifications & Purity: Free of DNA endonuclease and RNase.
• Stability And Storage: Store at -20℃.
• Storage Temp: Store at -20°C
• Shipped In: Ice chest + Ice pads

Product Description

One unit is the amount of enzyme required to catalyze the incorporation of 10nmol of deoxyribonucleotides (dNTPs) into a polynucleotide within 30 minutes at 37℃.Enzyme

Application：

Labeling of 5' overhangs; random priming labeling; Sanger dideoxy sequencing, etc.

Source：

E coli expressing the large fragment of DNA polymerase with mutations.

Enzyme storage buffer：

25mM Tris-HCl (pH7.5), 0.1mM EDTA , 1mM DTT, 50% (v/v) glycerol.

Reaction Buffer：

500mM Tris-HCl(pH8.0 at 25℃)，50mM MgCl2， 10mM DTT。缓冲液兼容性：在阿拉丁的内切酶反应缓冲液1X O、1X R、1X Y、2X Y中的活性为100%，在1X B、1X G中的活性为100%；在阿拉丁的Taq、Pfu DNA polymerase和M-MuLV反应缓冲液中的活性为100%。

Inactivation or inhibition：

Klenow Fragment can be inactivated by heating at 75℃ for 10 minutes or by adding appropriate amount of EDTA. Metal ion chelators, inorganic pyrophosphate (PPi), and large doses of inorganic phosphate (Pi) all have inhibitory effects on Klenow Fragment, Exo-.

Precautions：

Klenow Fragment, Exo- should be kept on ice when handling. Store at -20 ℃ immediately after use.This product is for R&D only. Not for drug, household, or other uses.For your safety and health, please wear a lab coat and disposable gloves during the operation.

Instructions for Use：

1. DNA labeling by random priminga. Set up the reaction in a microfuge tube on ice as follows:ComponentQuantityDNA10µl (100ng)Reaction Buffer (10X)5µl125µM Random Decamer or Hexamer10µlNuclease-free Waterto 40µlAfter mixing gently, boil for 5-10 minutes, and immediately cool on ice. Centrifuge to collect the droplets before proceeding to the next step.3 dNTP Mixture (0.25mM each , without the labeled dNTP)4µl[α-32P]-dNTP, ~110 TBq/mmol (3000Ci/mmol)1.85MBq (50µCi)Klenow Fragment, Exo-1µl (5U)Nuclease-free Waterto 50µlb. Mix well gently by pipetting or vortex. Centrifuge briefly to collect liquid at the bottom of the tube.c. For random decamer, incubate at 37℃ for 5 minutes; for random hexamer, incubate at 37℃ for 10 minutes.d. Add 4µl of 0.25mM dNTP, mix well and incubate at 37℃ for 5 minutes.e. Add 1µl of 0.5M EDTA, pH8.0 to stop the reaction.f. Take 1µl of the reaction mixture to determine the labeling efficiency.g. Purify the labeled probe with Sephadex G-50 or Bio-Gel P-60 or other appropriate kits.2. Labeling of 5' overhangs of dsDNAa. Set up the following reaction on ice.ComponentQuantityDigested DNA10~15µl (0.1~4µg)Reaction Buffer (10X)2µl[α-32P]-dNTP, ~15-30 TBq/mmol(400-800Ci/mmol)0.74 MBq (20µCi)or [α-32P]-dNTP, ~110 TBq/mmol(3000Ci/mmol)2.96 MBq (80µCi)3 dNTP Mixture (2.5mM each , without the labeled dNTP)2µlKlenow Fragment, Exo-0.2µl (1U)Nuclease-free Waterto 20µlb. Mix well gently by pipetting or vortex. Centrifuge briefly to collect liquid at the bottom of the tube.c. Incubate at 30℃ for 15 minutes.d. Incubate at 75℃ for 10 minutes to stop the reaction.3. For other uses, please refer to the protocol above or other relevant literature.