Eureka AIR delivers breakthrough ideas for toughest innovation challenges, trusted by R&D personnel around the world.

Method for constructing T vector based on terminal transferase activity of Taq enzyme

A terminal transferase and carrier technology, used in the introduction of foreign genetic material and recombinant DNA technology using a carrier, can solve the problems of high vector circularization background, low PCR product ligation efficiency, difficulty in screening positive clones, etc., and achieve convenient T-A cloning. , No self-cyclization background, simple and efficient method

Active Publication Date: 2014-08-13
CENT LAB FUJIAN ACADEMY OF AGRI SCI
View PDF0 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

During the T-A cloning process of PCR products, there will be problems such as high circularization background of the vector itself, low ligation efficiency of PCR products, and difficulty in screening positive clones.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Method for constructing T vector based on terminal transferase activity of Taq enzyme
  • Method for constructing T vector based on terminal transferase activity of Taq enzyme
  • Method for constructing T vector based on terminal transferase activity of Taq enzyme

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1: Preparation of linearized blunt-ended vector

[0022] 1. Departure Vector Linearization Reaction

[0023] With pBluescript SK(+) as the starting carrier, use Eco R V (Takara company) blunt-end endonuclease digested the pBluescript SK(+) vector, while ensuring that the endonuclease completely cuts the starting vector, and at the same time preventing the endonuclease from its own star activity, the ratio of the amount of vector added to the amount of enzyme was optimized as follows: 1 ug starting vector, add 10 U of enzyme.

[0024] Eco The R V enzyme digestion reaction system is:

[0025] 10×H buffer 10 μL

[0026] pBluescript SK(+) vector (50 ng / μL) 80 μL

[0027] Eco R V endonuclease (10 U / μL) 4 μL,

[0028] Add sterile distilled water to make up the volume to 100 μL. After reacting at 37°C for 2 h, 3 μL of the digested product was detected by 1% agarose gel electrophoresis.

[0029] 2. Test results

[0030] Such as figure 1 As shown, the ...

Embodiment 2

[0031] Embodiment 2: Intermediate T carrier preparation

[0032] 1. Linearized blunt-ended vector plus T reaction

[0033] To embodiment 1 gained Eco The RV digestion product was purified and recovered, and the concentration of the linearized blunt-end carrier was diluted to 50 ng / μL after recovery. use Taq DNA polymerase (Takara company) carries out adding T reaction to the linearized blunt-end carrier in the PTC-200 (Bio-Rad company) PCR instrument according to the following conditions, wherein the divalent metal cation that adds T is from MgCl 2 , MnCl 2 、CoCl 2 , CdCl 2 Among them, 1 mM concentration of CdCl is preferred 2 Add T metal cations for blunt-ended supports (see Table 1).

[0034] Table 1 T addition efficiency of four kinds of metal cations with different concentrations

[0035]

[0036] Taq DNA polymerase plus T reaction system is:

[0037] 10×PCR Buffer (Mg 2+ free) 10 μL

[0038] Linearized blunt-ended vector (50 ng / μL) 55 μL

[0039] dTTP (...

Embodiment 3

[0062] Embodiment 3: T carrier preparation

[0063] 1. The middle T vector removes the 3′-end without T vector

[0064] Use T4 DNA ligase (Fermentas company) to perform ligation reaction on the PTC-200 (Bio-Rad company) PCR instrument for the intermediate T vector obtained in Example 2 according to the following conditions.

[0065] T4 DNA Ligase ligation reaction system is:

[0066] 10×T4 DNA Ligase Buffer 5 μL

[0067] Intermediate T carrier 30 μL (about 2~3 μg)

[0068] T4 DNA ligase (5 U / μL) 3 μL,

[0069] Add sterilized distilled water to make up the volume to 50 μL, mix well and incubate at 16 °C for 5 h on a PTC-200 (Bio-Rad Company) PCR instrument. After the ligation reaction, the carrier without T at the 3′-end will re-form a supercoil under the action of T4 DNA Ligase, while the carrier with T at the 3′-end will maintain a linear state. The reaction product is electrophoresed on a 1% agarose gel Separation, gel cutting and recovery of DNA fragments at 3000 bp...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention relates to a method for vector construction in the field of gene engineering, in particular to a method for constructing a T vector based on the terminal transferase activity of a TaqDNA polymerase. The method comprises the steps of adding T to the 3'-terminal of a linearized blunt end starting vector mainly by using the terminal transferase activity of the TaqDNA polymerase, and then removing the self cyclization background of the 3'-terminal T-free vector by using a T4DNA ligase to obtain a linear T vector having a single protruding T at the 3'- terminal. The T vector obtained by the method is stable in quality, low in self cyclization background and good in cloning effect, is suitable for small-scale preparation and large-scale production, and will play an important role in the field of gene engineering.

Description

technical field [0001] The invention relates to a method for vector construction in the field of genetic engineering, specifically a method based on Taq A method for constructing a T carrier with DNA polymerase terminal transferase activity. Background technique [0002] T vector is a convenient linear vector for cloning PCR products, and T-A cloning technology has been proved to be extremely valuable for cloning PCR products. The principle of this method is to use the Taq DNA polymerase has a non-template-dependent terminal transferase activity, which can add an overhanging dA base to the 3'-end of each strand of the PCR double-stranded product, and the 3'-end of the linear T vector just contains it. The protruding dT bases of complementary pairing, so as to realize the T-A complementary connection. [0003] Currently, there are two classic methods for constructing T vectors. The first is to pass the plasmid through the Eco R V or Sam Restriction endonuclease lin...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12N15/63C12N15/64
Inventor 吕新李玥仁陈丽华刘兰英李巍
Owner CENT LAB FUJIAN ACADEMY OF AGRI SCI
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com