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T vector construction method

A vector and linearized vector technology, applied in the field of genetic engineering, can solve the problems of unstable quality, high price, difficulty in popularization, etc., and achieve the effects of simple and efficient method, low self-cyclization rate, and high cloning effect.

Active Publication Date: 2012-11-21
中生方政生物技术股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since adding T at the 3' end is a non-preferentially polymerized nucleotide, only part of the 3' end can be added with T, or more than one T can be added, resulting in a high circularization rate of the vector itself during the cloning process, and the connection is unstable. Increased difficulty for positive clones
In addition, the steps of the T carrier produced by this method are relatively cumbersome. The output of each T carrier is limited, the quality is unstable, and the quality difference between batches is large. It is not suitable for large-scale production, and because it is expensive, it cannot be recycled. must be difficult

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0041] Embodiment 1: the preparation of Xcm I box

[0042] Using the pUC19 plasmid as a template, the upstream primer having the nucleotide sequence shown in SEQ ID NO: 2 and the downstream primer having the nucleotide sequence shown in SEQ ID NO: 3 were amplified to perform PCR amplification.

[0043] The PCR amplification reaction system is:

[0044] 10×taq buffer (with 1.5mM Mg 2+ ) 5μl

[0045] 10mM dNTP 1μl

[0046] 1μl of 10μM upstream primer

[0047] 10μM downstream primer 1μl

[0048] pUC19 plasmid (20ng / μl) 1μl

[0049] Taq enzyme (5U / μl) 0.5μl

[0050] Add sterilized distilled water to make up the total amount of the system to 50 μl.

[0051] The PCR reaction procedure is:

[0052] Pre-denaturation at 94°C for 3 minutes

[0053]

[0054] Extend at 72°C for 10min

[0055] The resulting PCR product was taken in 5 μl and detected by 1% agarose gel electrophoresis. The results are shown in figure 1 .

Embodiment 2

[0056] Embodiment 2: the preparation of former T carrier

[0057] The PCR product obtained in Example 1 was purified and recovered, and the recovered product was respectively combined with pGEM-T Easy Vector (Promega Company), pBS-T Vector (original Ping Hao biology), pSURE-T Vector (original Ping Hao biology) in T4DNA ligase The ligation was carried out under the effect of ligation, and the ligation product was transformed into TOP10 competent cells, and the transformed cells were plated on LB solid medium containing ampicillin for selection. The plasmids were extracted from the positive clones screened, and the plasmids containing about 500 bp target fragments identified by enzyme digestion were the pre-T vectors.

Embodiment 3

[0058] Embodiment 3: Preparation of T carrier

[0059] With Xcm I restriction endonuclease embodiment 2 gained former T carrier, the result sees figure 2 . The sizes of the vectors pGEM-T, pBS-T and pSURE-T are 3037bp, 3022bp and 2773bp respectively, so the band around 3000bp is the linearized vector. The linearized vectors were recovered to obtain T vectors pGEM-T 1, pBS-T 1, and pSURE-T 1.

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Abstract

The invention relates to the technical field of gene engineering and discloses a T vector construction method which comprises the following steps: using a pGEM-T Easy Vector, a pBS-T Vector and a pSURE-T Vector as starting vectors to be connected with an Xcm I cassette to obtain a T vector precursor; and then, using Xcm I restriction enzymes to cut the T vector precursor, and recovering the linearized vector to obtain the T vector, wherein the sequence of the Xcm I cassette comprises a spacer DNA sequence and additional sequences closely connected to both sides of the spacer DNA sequence respectively, and the additional sequence comprises an Xcm I enzyme cutting site sequence and three protective basic groups at the 5' end of the Xcm I enzyme cutting site sequence. The T vector construction method has the advantages of simple operation, high efficiency, stable quality of the obtained T vector, low self cyclization rate and high cloning effect, and can avoid frame shift in the gene expression process of the cDNA sequences of (3n+2) numbered nucleotides.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a method for constructing a T vector. Background technique [0002] PCR is a basic technique in molecular biology and genetic engineering research, and it is widely used in vitro to amplify and isolate known or unknown specific DNA fragments. Rapid and effective cloning of PCR products into plasmid vectors is a prerequisite for high-quality sequencing, in vitro transcription, and in vitro translation experiments to achieve multiple uses of PCR products. Usually, PCR products do not have sticky ends and can only be cloned by blunt end ligation. Since Taq DNA polymerase has a template-independent terminal transferase activity, during PCR amplification, the 3′ end of the PCR product will be in a template-independent manner. Add an adenine residue (A). As a convenient PCR product cloning carrier, the T vector has a protruding thymus nucleotide (T) at the 3' end, which c...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/66
Inventor 李艳萍
Owner 中生方政生物技术股份有限公司