Process for preparing T vector

A vector and endonuclease technology, applied in the direction of using vectors to introduce foreign genetic material, recombinant DNA technology, etc., can solve the problems of reducing the quality and instability of T vectors

Inactive Publication Date: 2004-03-17
SUN YAT SEN UNIV
View PDF0 Cites 5 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the production of this type of T vector, in order to reduce the background and improve the cloning efficiency, overdigestion is a commonly used strategy, which makes the impact of residual exonuclease activity on the target T vector more significant. Thereby significantly reducing the quality of the resulting T carrier and making it unstable

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
  • Process for preparing T vector
  • Process for preparing T vector
  • Process for preparing T vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0014] Embodiment 1. Construction of plasmid pUC-HX (pre-T vector)

[0015] According to the sequence shown in Figure 1, the HphI-XcmI linker sequence was synthesized by conventional methods. The two strands of the linker were synthesized separately, then dissolved in double-distilled water, mixed equimolarly, and annealed at 37°C for 15 minutes to obtain a double-stranded HphI-XcmI linker with a full length of 60bp (such as figure 1 shown). The linker contains two concatemers of HphI and XcmI recognition sequences arranged in an inverted repeat manner GGTG A (NNNNT / NNNN) (The first base upstream of the cut site 5' of the endonuclease XcmI in the sequence is set as T), and the endonuclease XcmI digests the vector containing the linker to obtain a mature T vector.

[0016] The DNA linker HphI-XcmI synthesized above was inserted into the EcoRI+SalI window of the plasmid pUC18, and after ligation and transformation, the recombinant plasmid pUC-HX was obtained, and its rest...

Embodiment 2

[0017] The synthesis of embodiment 2.ECS

[0018] Two exonuclease competitive substrates ECS1 and ECS2 were synthesized according to conventional methods, and their specific sequences were 5'p-CCAGCGCTGGT and 5'-CAAGAATTCATGGAATTTGGCAACCTA, respectively. ECS1 is an oligo DNA molecule with a palindromic structure of 11 nucleotides containing a 5' phosphate group, which can pair with each other in a 37°C overdigestion system to form a double-stranded structure with DNA ends similar to T vectors. ECS2 is a common PCR primer of 27 nucleotides (without 5' phosphate group), which can maintain a single-stranded structure in the overdigestion system at 37°C.

Embodiment 3

[0019] Example 3. Acquisition of carrier pUC-HX-T (T carrier)

[0020] In the three cases of adding ECS1 and ECS2 and not adding exonuclease competitive substrates, respectively, the vector pUC-HX was digested with restriction endonuclease XcmI to obtain the mature T vector pUC-HX-T.

[0021] The enzyme digestion reaction system is as follows:

[0022] pUC-HX 2 μg

[0023] Exonuclease competitive substrate (ECS1 / ECS2) 2μg / NULL

[0024] XcmI 15U

[0025] Buffer 2 (NEB) 10μl

[0026] wxya 2 O to a final volume of 100 μl

[0027] React at 37°C for 2 hours, phenol extraction will inactivate the enzyme. The digested product was run on 1.0% agarose gel electrophoresis, and a DNA fragment with a size of about 2.7 kb was recovered from the gel (refer to the QIAGEN gel recovery kit manual), which was the vector pUC-HX-T.

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

A process for preparing T vector by using endoenzyme to digest the pre-T vector features that the single- or dual-chain oligo-DNA as the competitive substrate of exoenzyme, whose mole concentration is at least 10 times higher than that of pre-T vector, is added to the over-digestive system used to prepare said T vector in the condition of no excessive increasement of total DNA concentration to suppress the influence of exoenzyme activity on T vector, so obtaining excellent T vector with stable quality.

Description

technical field [0001] The invention relates to a preparation method of a T vector, in particular to a method for obtaining a T vector by digesting a pre-T vector with an endonuclease. Background technique [0002] T / A” cloning method (Clark, J.M.Novel non-templated nucleotide addition reactions catalyzed byprocaryotic and eucaryotic DNA polymerases. Nucleic Acids Res.1988, 16, 9677-86; Holton, T.A.andGraham, M.W.A simple and efficient method for direct cloning of PCR products using ddT-tailed vectors. Nucleic Acids Res.1991, 19, 1156; Marchuk, D., Drumm, M., Saulino, A. and Collins, F.S. Construction of T-vectors, a rapid and general system for direct cloning of unmodified PCR products. Nucleic Acidds Res. 1991, 19, 1154; Mead, D.A., Pey, N.K., Herrnstadt, C., Marcil, R.A. and Smith, L.M.A universal method for the direct cloning of PCR amplified nucleic acid. Biotechnology (NY). 1991,9,657-63) is a kind of cloning method that is develope...

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
Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/09C12N15/64
Inventor 王珣章贺雄雷付捷龙綮新
Owner SUN YAT SEN UNIV
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
Try Eureka
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap