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A kind of multifunctional cloning carrier and its application method

A cloning vector and multi-functional technology, which can be used in the introduction of foreign genetic material using vectors, recombinant DNA technology, etc., can solve the problems of cumbersome steps and long time, and achieve the effect of low experimental cost, few experimental operation links, and improved scientific research efficiency.

Active Publication Date: 2016-04-27
湖南赛哲智造科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method is cumbersome and time-consuming, and is limited by gene sequences and restriction endonuclease recognition sequences.

Method used

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  • A kind of multifunctional cloning carrier and its application method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] A kind of multifunctional cloning vector, its construction steps are as follows:

[0044] 1) Design a pair of primers with 5'-terminal phosphorylated sequences as follows:

[0045] F:5'-CTGTCAGACCAAGTTTACTCATA-3'

[0046] R:5'-TACCAATGCTTAATCAGTGAGGC-3'

[0047] The first base at the 5' end of the primer is A or G.

[0048] 2) PCR amplification

[0049] Using the PUC19 plasmid as a template, a high-fidelity enzyme PCR amplification was used to obtain a sufficient amount of product, and then the residual template plasmid was digested with DpnI endonuclease, and the product obtained by gel purification was a multifunctional cloning vector.

[0050] The PCR reaction system and conditions described therein are as follows:

[0051] The reaction system was designed as a total system of 50 μl, specifically 6 μl of 5×PCR Buffer (buffer), 2 μl of dNTPmix (deoxyribonucleoside triphosphate mixture) with a concentration of 2.5 mM, 1 μl of upstream and downstream primers with a ...

Embodiment 2

[0057] How to use the carrier:

[0058] 1 Primer Design Requirements

[0059] This vector has a preference for bases. When designing primers, make sure that the first base at the 5' end of the primers is A or G;

[0060] 2 The establishment of the reaction system

[0061] Add various components to a 0.2ml PCR tube according to the following reaction system:

[0062]

[0063] Gently flick the reaction tube to mix the contents and centrifuge briefly for 3-5 seconds;

[0064] 3 Put the mixed reaction solution at room temperature (22°C-30°C) to react for 5 minutes. After the reaction, put the reaction tube on ice and carry out the subsequent conversion reaction. If the length of the inserted fragment is greater than 2kb, the reaction time can be extended to 30 minutes;

[0065] 4 Conversion

[0066] 1) Take part of the ligation product and add it to 50-100 μl of competent cells (the competent cells just taken out of the refrigerator are placed in an ice bath for about 20 min...

Embodiment 3

[0072] Positive clone detection

[0073] a Rapid colony PCR detection

[0074] Pick colonies for direct PCR detection, and identify positive clones by PCR according to the following reaction conditions: pre-denaturation at 95°C for 3 min, denaturation at 94°C for 30s in the cycle, annealing at 55°C for 30s, 25 cycles, and extension at 72°C for Xs (1K / min) , Continue to extend at 72°C for 5min after PCR reaction cycle, and then store at 4°C;

[0075] b Sequencing identification: The DNA sequence is determined after the preliminary identification by the rapid colony method; the sequences of the PCR identification and sequencing primers of this vector are as follows:

[0076] F5'-AGACAGATCGCTGAGATAGG-3'

[0077] R5'-CGTTAAGGGATTTTGGTCATGAG-3'

[0078] The positive clones are more than 90%, the false positive rate is low, the experimental cost is low, and the experimental operation links are less than the traditional cloning method, which is convenient and fast.

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Abstract

The present invention discloses a multifunctional cloning vector, wherein the construction method comprises adopting a resistance gene expression self-saving manner and directly adopting the conventional antibiotic to screen positive clones, and specifically comprises: designing a pair of primer sequences with 5' terminal phosphorylation, adopting PUC19 plasmid as a template, and adopting high-fidelity PCR amplification to obtain the multifunctional cloning vector. With the vector prepared by using the method, various PCR products can be efficiently cloned, efficient cloning efficiency of the blunt-ended PCR product and the general Taq enzyme amplification product can be achieved, and the vector of the present invention can be adopted to completely replace the blunt-ended cloning vector and the TA cloning T vector. The method for preparing the multifunctional cloning vector has advantages of general positive rate of greater than 90%, low false positive rate, low test cost, less experiment operation steps, convenience and rapidness. The invention further discloses the use method of the multifunctional cloning vector.

Description

technical field [0001] The invention relates to a multifunctional cloning vector, and also relates to a method for constructing and using the multifunctional cloning vector, belonging to the technical field of cloning in genetic engineering. Background technique [0002] With the development of large-scale sequencing technology, the genome sequences of more and more species have been determined. In order to further study the function of unknown genes, it is necessary to clone the studied genes to provide the possibility for further research. Therefore, the application of efficient gene cloning technology is particularly important. Conventional gene cloning technology, using primers containing restriction endonuclease recognition sequences to amplify the target gene by polymerase chain reaction (PCR), and the obtained PCR amplification products are subjected to corresponding restriction endonucleases processed, and ligated into the corresponding restriction endonuclease-trea...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/70C12N15/64
Inventor 张茂雷何东海郝晓燕康涛
Owner 湖南赛哲智造科技有限公司
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