Multifunctional cloning vector and use method thereof

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 carrier, and also relates to a construction method and a use method of the multifunctional cloning carrier, 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 functions 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 uses primers containing restriction endonuclease recognition sequences to amplify the target gene through polymerase chain reaction (PCR), and the obtained PCR amplification products are subjected to corresponding restriction endonuclease treatment, and connected to the vector treated with the correspo...

Examples

Embodiment 1

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

[0044] 1) Design a pair of primers with 5' end phosphorylation 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, high-fidelity enzyme PCR was used to amplify a sufficient amount of the product, and then the remaining template plasmid was digested with DpnI endonuclease, and the product obtained by gel purification was the multifunctional cloning vector.

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

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

Embodiment 2

[0057] How to use the carrier:

[0058] 1 Primer Design Requirements

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

[0060] 2. Establishment of reaction system

[0061] Add various components into a 0.2ml PCR tube according to the reaction system in the table below:

[0062]

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

[0064] 3 Place the mixed reaction solution at room temperature (22°C-30°C) for 5 minutes. After the reaction, place the reaction tube on ice for the subsequent transformation reaction. If the length of the insert is greater than 2kb, the reaction time can be extended to 30 minutes;

[0065] 4 transformations

[0066] 1) Take part of the ligation product and add it to 50-100 μl competent cells (thaw the competent cell just taken out of the refrigerator on an ice bath for about 20 minutes, add the ligation prod...

Embodiment 3

[0072] positive clone detection

[0073] aRapid colony PCR detection

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

[0075] b Sequencing and identification: the determination of the DNA sequence after preliminary identification by the rapid colony method; each identification and sequencing primer sequence of the carrier PCR is 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 is less than the traditional cloning method, which is convenient and fa...

technical field

[0001] The invention relates to a multifunctional cloning carrier, and also relates to a construction method and a use method of the multifunctional cloning carrier, 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 functions 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 uses primers containing restriction endonuclease recognition sequences to amplify the target gene through polymerase chain reaction (PCR), and the obtained PCR amplification products are subjected to corresponding restriction endonuclease treatment, and connected to the vector treated with the correspo...

Examples

Embodiment 1

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

[0044] 1) Design a pair of primers with 5' end phosphorylation 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, high-fidelity enzyme PCR was used to amplify a sufficient amount of the product, and then the remaining template plasmid was digested with DpnI endonuclease, and the product obtained by gel purification was the multifunctional cloning vector.

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

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

Embodiment 2

[0057] How to use the carrier:

[0058] 1 Primer Design Requirements

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

[0060] 2. Establishment of reaction system

[0061] Add various components into a 0.2ml PCR tube according to the reaction system in the table below:

[0062]

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

[0064] 3 Place the mixed reaction solution at room temperature (22°C-30°C) for 5 minutes. After the reaction, place the reaction tube on ice for the subsequent transformation reaction. If the length of the insert is greater than 2kb, the reaction time can be extended to 30 minutes;

[0065] 4 transformations

[0066] 1) Take part of the ligation product and add it to 50-100 μl competent cells (thaw the competent cell just taken out of the refrigerator on an ice bath for about 20 minutes, add the ligation prod...

Embodiment 3

[0072] positive clone detection

[0073] aRapid colony PCR detection

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

[0075] b Sequencing and identification: the determination of the DNA sequence after preliminary identification by the rapid colony method; each identification and sequencing primer sequence of the carrier PCR is 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 is less than the traditional cloning method, which is convenient and fa...

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