Preparation method of T vector for T-A cloning

A system, T-A technology, applied in the direction of using vectors to introduce foreign genetic material, recombinant DNA technology, etc., can solve the problems of difficult quality control, expensive endonucleases, and high background of non-recombinant transformants

Active Publication Date: 2006-07-12
生工生物工程(上海)股份有限公司
View PDF0 Cites 4 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Now due to the strict quality control of the manufacturer, its efficiency has also been greatly improved, and it can meet the routine cloning operation. However, due to its many operation steps, the quality is not easy to control. At present, it is only used in several common vectors, and it must be performed by a professional company. to produce, so the price is more expensive
The third method is currently also used in the preparation of T vectors, but there is a potential problem in the construction of T vectors with endonucleases. Partially digested pre-T vectors are mixed in T vectors, which will lead to e

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
  • Preparation method of T vector for T-A cloning

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0020] Example 1: Introduce the preparation method of T vector for T-A cloning, such as figure 1 :

[0021] Choose a high copy number commonly used plasmid, such as pBR322 series plasmids, pUC series plasmids, etc.

[0022] In this example, the pZErO-2 plasmid was selected as the template.

[0023] 1. Using the pZErO-2 plasmid as a template, according to its DNA sequence, four pairs of primers were designed, and two mutant plasmids with differences in specific sequences were constructed through site-directed mutagenesis. The two new plasmids were named pNTv1 and pNTv2 respectively. Among them, a new restriction site BamHI and a TTTTAAA sequence box that can be recognized by DraI endonuclease are introduced into pNTv1, as shown in Figure 1: The dotted line represents the restriction site BamHI, and the I and arrow symbols in the figure represent the TTTTAAA sequence box , Can be cut by DraI endonuclease; pNTv2 introduces a new restriction site HindIII and a TTTAAAA sequence box that ...

Example Embodiment

[0029] Example 2: Performance detection of T vector pNTv

[0030] 1. The recovered mixture of pNTv3 and pNTv4 is mixed with DNA with 3'dA in three different molar ratios of 1:3, 1:5, and 1:7, and T4 DNA ligase is added, and ligated at 16°C.

[0031] 2. The connected product electrotransforms TOP 10 competent cells.

[0032] 3. The transformed product was coated on LB plate and incubated at 37°C.

[0033] 4. The colony was screened by PCR and the plasmids were extracted and identified by restriction enzyme digestion. The results showed that the recombination efficiency reached more than 90%.

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 discloses a method for preparing for T carrier used in T-A clone, which chooses a normal plasmid with high copy quantity as table to design four pairs of primer and uses point discontinuity to construct two types of differential discretion plasmid in the order advance sequence; it separately constructs TTTTAAA and TTTAAAA sequence box on the polyclonal point of the two types of discretion plasmid and leads two different enzyme cutting points BamHI and HindIII; it separately leads the discretion plasmids in the bacteria to augment and uses the inner cutting enzyme of the identifying TTTAAA sequence to cut, mix, degenerate and compound the augmented plasmid; it dose enzyme cutting to the reacted mixture, dispels the parental generation molecule to separate the T carrier. It adopts special biology compositing technique to produce the T carrier.

Description

technical field [0001] The invention relates to a method for constructing a plasmid vector in the field of genetic engineering, in particular to a method for preparing a T vector for T-A cloning by using a unique biosynthesis method. Background technique [0002] T-A cloning method (Clark, J.M.Novel non-templated nucleotide addition reactions catalyzed by procaryotic and eucaryotic DNA polymerases (new prokaryotic or eukaryotic DNA polymerase catalyzed non-template-dependent nucleic acid addition reactions). Nucleic Acids Res.1988, 16, 9677-9686; 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 product (construction of T-vectors, a rapid and general A system for direct cloning of original PCR products). Nucleic Acids Res.1991, 19, 1154; Holton, T.A.and Graham, M.W.A simple and efficient method for direct cloning of PCR products using ddT-tailed vectors (a di...

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): C12N15/63C12N15/66
Inventor 田大成张远莉王强金欣庆丁静
Owner 生工生物工程(上海)股份有限公司
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