Unlock instant, AI-driven research and patent intelligence for your innovation.

T vector and its construction method and pre-T vector

A carrier and promoter technology, applied in the field of genetic engineering, can solve the problems of hybridization and high background of non-recombinant transformants

Inactive Publication Date: 2008-05-21
CHINA AGRI UNIV
View PDF0 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is a potential problem whether XcmI or AhdI and its isoschizase are used to construct T vectors: partially digested pre-T vectors mixed in T vectors will lead to excessive background of non-recombinant transformants (Mead, D.A., Pey, N.K., Herrnstadt, C., Marcil, R.A., Smith, L.M., 1991. A universal method for the direct cloning of PCR amplified nucleic acid. Bio / Technology 9, 657-663; Harrison, J., Molly, P.L., Clark, S.J., 1994. Direct cioning of polymerase chain reaction products in an XcmI T-vector. Anal. Biochem. 216, 235-236)
However, a new problem brought about by the introduction of spacer DNA is that since circular plasmids move faster in agarose gel electrophoresis than linear plasmids of comparable molecular weight, undigested circular plasmids (pre-T vector, with spacer DNA) is often mixed with the T vector (without spacer DNA) whose molecular weight is smaller than itself, and finally causes the background of non-recombinant transformants to be too high

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
  • T vector and its construction method and pre-T vector
  • T vector and its construction method and pre-T vector
  • T vector and its construction method and pre-T vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Embodiment 1, construct the former T carrier of Gen resistance

[0060] 1. Construct the intermediate vector of Gen resistance

[0061]1) Use the pEZ-TNL vector (http: / / deepgreen.stanford.edu / cell imaging site / html / vectors.html) as a template and GenF and GenR as primers to amplify the Gen gene sequence. The PCR amplified product was recovered after agarose gel electrophoresis, and then digested with BspHI+SmaI. The PCR primers are as follows:

[0062] GenF: 5'-AAAA GACGCACACCGTGGAAACGGATGAAGGCAC-3' (the base sequence in the box is the BspHI restriction site); GenR: 5'-AAAA CGGCGTTGTGACAATTTACCGAACAACTCC-3' (the base sequence in the box is the SmaI restriction site). The reaction system of PCR amplification is: 5U Pfu, 0.2mmol / L dNTPs, 1×Pfu buffer in 50μL reaction system, 10μmol / L of primers, 5ng of template; the cycle program of PCR amplification is: pre-denaturation at 94℃ for 5min, (94°C, 30S; 60°C, 30S; 72°C, 2min) × 30, 72°C for a final extension of 5min. ...

Embodiment 2

[0090] Embodiment 2, construct the former T carrier of Amp resistance

[0091] 1. Remove the AhdI restriction site on the pBlueScript SK(-) vector

[0092] Introduce mutated bases into the PCR primers, use pBluescript II SK(-) carrier (GenBankAccession No.X52330) as template, and use Pfu DNA polymerase to perform PCR amplification, and then perform agarose gel electrophoresis and then cut the gel to recover the PCR amplification product, establish a ligation reaction system, and ligate at 4° C. for 16 hours to circularize the PCR amplification product (linear vector). Among them, the reaction system of PCR amplification is: 5U Pfu, 0.2mmol / L dNTPs, 1×Pfu buffer in 50μL reaction system, 10μmol / L of primers, and about 5ng of template; the cycle program of PCR amplification is: Denaturation for 5min, (94°C, 30S; 60°C, 30S; 72°C, 5min) × 30, 72°C for a final extension of 5min. Ligation reaction system: 10 μL ligation system containing 3U ligase (Promega), 1× ligation buffer, 50n...

Embodiment 3

[0102] Example 3. Construction of Kan-resistant pre-T vectors

[0103] pGWGENO1A, pGWGENO1B and pGWGENO1C were respectively digested with NheI and BspHI, and three small fragments were recovered by cutting the products after agarose gel electrophoresis. The pENTR1A plasmid was also digested with NheI and BspHI, and the digested product was subjected to agarose gel electrophoresis, and then the gel was cut to recover a large fragment. The three small fragments and large fragments were ligated at 4°C for 16 hours. Among them, 10 μL ligation system contains 3U ligase (Promega), 1× ligation buffer, about 100 ng of large fragments, and about 50 ng of small fragments. The ligation product was transformed into Escherichia coli DB3.1 strain, and positive clones were screened on the Kan resistance plate. Positive clones were identified by AhdI digestion. The corresponding plasmids are the pre-T vectors pGWKAN01A, pGWKAN01B and pGWKAN01C with Kan resistance.

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 present invention discloses T-vector, and its construction process and pre-T-vector. The T-vector is linear double strand DNA plasmid with single protruding T terminus in the 3' ends, attL1 sequence connected to the inside of one 3' protruding T terminus and attL2 sequence connected to the inside of one other 3' protruding T terminus. Its preparation process includes the following steps: 1) preparing pre-T-vector containing attL1-attL2 box, which includes AhdI box, attL1 sequence in the upstream of the AhdI box and attL2 sequence in the downstream of the AhdI box, with the AhdI box including interval DNA sequence and two AhdI cleavage site sequences connected closely on two sides of the interval DNA sequence separately; and 2) cleaving the pre-T-vector with AhdI or its isoschizomer to obtain T-vector. The T-vector may be used as the primer vector.

Description

technical field [0001] The invention relates to a T vector and its construction method and its pre-T vector in the field of genetic engineering. Background technique [0002] λ bacteriophage integrates into the bacterial chromosome by a set of site-specific recombination system and completes the conversion between lytic cycle and lysogenic cycle (Bushman W, Thompson J F, Vargas L, et al. Control of directionality in lambda site specific recombination. Science , 1985, 230: 906-911; Landy A. Dynamic, structural, and regulatory aspects of lambdasite-specific recombination. Ann Rev Biochem, 1989, 58: 913-949). Invitrogen has developed a set of technology for in vitro DNA recombination based on the site-specific recombination system of λ phage, called Gateway cloning technology. Gateway technology is a general cloning method, which can quickly and efficiently construct the target DNA sequence into a variety of vector systems compatible with Gateway technology for functional anal...

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): C12N15/63C12N15/11
Inventor 陈其军王学臣陈珈
Owner CHINA AGRI UNIV