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Kit for completing DNA assembly by depending on T5 exonuclease and PEG8000 and application thereof

A technology of exonuclease and kit, which is applied in the biological field to achieve the effects of low cost, improved efficiency, and convenient operation

Active Publication Date: 2018-11-20
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

After searching, by constructing a method TEDA (named TEDA assembly, which relies on a small amount of T5 exonuclease, with the help of PEG8000 and optimized conditions to complete DNA assembly) T 5 E xonuclease D dependent A assembly), and related kits that rely on T5 exonuclease and PEG8000 to complete DNA assembly have not been reported

Method used

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  • Kit for completing DNA assembly by depending on T5 exonuclease and PEG8000 and application thereof
  • Kit for completing DNA assembly by depending on T5 exonuclease and PEG8000 and application thereof
  • Kit for completing DNA assembly by depending on T5 exonuclease and PEG8000 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1. A kit that relies on T5 exonuclease and PEG8000 to complete DNA assembly

[0065] The kit consists of T5 exonuclease and a buffer system containing PEG8000; based on the total system of 20uL, the amount of T5 exonuclease is 0.16U / total reaction system, and the buffer system components of PEG8000 are 115mM, pH=7.5 Tris-HCl, 11 mM MgCl 2 , 11 mM DTT and 6% PEG8000 by mass.

[0066]Among them, each component in the above-mentioned kit relying on T5 exonuclease and PEG8000 to complete DNA assembly is allowed to be pre-configured as 4 / 3×, 2× or 5× concentration concentration, which is reserved for the later addition of DNA, and the concentration conditions Does not affect the effect of the assembly reaction. The T5 exonuclease and reaction buffer in the kit are pre-mixed or post-added.

[0067] The above kit has been tested and stored at -20°C and -80°C without affecting the reaction effect; and the kit can be stored at low temperature for more than half a year...

Embodiment 2

[0068] Example 2. A kit that relies on T5 exonuclease and PEG8000 to complete DNA assembly

[0069] The kit consists of T5 exonuclease and a buffer system containing PEG8000; the kit is based on a total system of 20uL, the amount of T5 exonuclease is 0.04U / total reaction system, and the buffer system component of PEG8000 is 105mM , Tris-HCl at pH=7.5, 10 mM MgCl 2 , 10mM DTT and 5% PEG8000 by mass.

[0070] Among them, each component in the above-mentioned kit relying on T5 exonuclease and PEG8000 to complete DNA assembly is allowed to be pre-configured as 4 / 3×, 2× or 5× concentration concentration, which is reserved for the later addition of DNA, and the concentration conditions Does not affect the effect of the assembly reaction. The T5 exonuclease and reaction buffer in the kit are pre-mixed or post-added.

[0071] The above kit has been tested and stored at -20°C and -80°C without affecting the reaction effect; and the kit can be stored at low temperature for more than ...

Embodiment 3

[0072] Example 3. A kit that relies on T5 exonuclease and PEG8000 to complete DNA assembly

[0073] The kit consists of T5 exonuclease and a buffer system containing PEG8000; based on the total system of 20uL, the amount of T5 exonuclease is 0.02U / total reaction system, and the buffer system components of PEG8000 are 110mM, pH=7.5 Tris-HCl, 9mM MgCl 2 , 9mM DTT and PEG8000 with a mass ratio of 4%.

[0074] Among them, each component in the above-mentioned kit relying on T5 exonuclease and PEG8000 to complete DNA assembly is allowed to be pre-configured as 4 / 3×, 2× or 5× concentration concentration, which is reserved for the later addition of DNA, and the concentration conditions Does not affect the effect of the assembly reaction. The T5 exonuclease and reaction buffer in the kit are pre-mixed or post-added.

[0075] The above kit has been tested and stored at -20°C and -80°C without affecting the reaction effect; and the kit can be stored at low temperature for more than h...

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Abstract

The invention discloses a kit for completing DNA assembly by depending on T5 exonuclease and PEG8000 and application thereof. The kit consists of the T5 exonuclease and a buffer system containing thePEG8000; in terms of a total system of 20 microliters, the using amount of the T5 exonuclease is 0.02 U to 0.16 U in a total reaction system, and the buffer system containing the PEG8000 comprises thecompositions of 110+ / -5 mM Tris-HCl with the pH of 7.5, 10+ / -1 mM MgCl2, 10+ / -1 mM DTT and PEG8000 with the mass ratio of 5+ / -1%. The invention also discloses the application of the kit in DNA recombination seamless cloning. Experiments prove that the kit and a related DNA assembling method are of accurate, efficient and convenient characteristics which are the same as those of a commercialized assembling reagent, but the selling price of the kit is 100 times lower than that of a commercialized kit, and the application is more economical and practicable by compared with an existing method.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a DNA assembly kit and its application, in particular to a cheap kit relying on T5 exonuclease and PEG8000 to complete DNA assembly and its application. Background technique [0002] DNA cloning is a technology invented 40 years ago. The fusion of DNA molecules is completed by enzyme cutting and ligation, which is a milestone in the development of molecular biology. It is still the most widely known method. However, with the emergence and development of metabolic engineering and synthetic biology, the construction and modification of genetic modules have higher and higher requirements for cloning methods. The inefficiency, sequence dependence and non-patterning of enzyme cutting and ligation methods make it unsuitable for current operations. In recent years, a variety of new technologies for DNA assembly have emerged to overcome the limitations of traditional enzyme cutting and ligatio...

Claims

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Application Information

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IPC IPC(8): C12P19/34C12P19/20C12N15/70
CPCC12N15/70C12P19/20C12P19/34
Inventor 夏永振荀鲁盈谷立川
Owner SHANDONG UNIV
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