Expression type pre-T vector, preparation thereof and applications

A carrier and ribosome technology, applied in the field of expressive pre-T vectors, can solve the problems of low quality, expensive and difficult to control enzyme-digested plasmid DNA, and achieve the goals of improving the success rate of sequencing, high efficiency of enzyme digestion, and saving money Effect

Inactive Publication Date: 2008-12-31
ZHEJIANG UNIV OF TECH
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  • Abstract
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  • Claims
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AI Technical Summary

Problems solved by technology

This method has obvious disadvantages: first, it cannot guarantee that 100% of the ends of the carrier molecules can be added with dT, and second, it is difficult to control exactly 1 dT to be added.
[0013] The following problems may exist in the preparation of T vectors by enzymatic methods: First, if the quality of the digested plasmid DNA is not high, the foreign gene cannot be inserted into the vector, and the vector can grow on the resistant plate after transforming competent cells
This method requires the use of expensive X-gal, and it is troublesome to prepare blue-white screening plates
Second, if the spatial position of the two XcmI restriction sites is relatively close, it will affect the restriction efficiency of the vector. Even if the purity of the carrier DNA is high and the quality of the enzyme is also good, only one restriction site will be digested In some cases, this linear vector cannot be ligated with the PCR product, but the vector is self-ligated, resulting in the generation of negative clones

Method used

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  • Expression type pre-T vector, preparation thereof and applications

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1: Amplification of DNA sequence containing tryptophanase promoter

[0038] Primer:

[0039] MK1: 5’-TATGGATCCTGCTCCCCGAACGATTGTGA-3’

[0040] MK2:

[0041] 5’-TAGTTAGGCCCATTATACCCTTGGTCCTTCATTTATTTTAATTACAGTGA-3’ (modified MK2)

[0042] Template DNA: E. coli K-12 cells

[0043] PCR reaction system and its parameters:

[0044] 10×Buffer(with Mg 2+ ) 5μL

[0045] 10mmol / L each dNTPs 2μL

[0046] 10mmol / L MK1 2μL

[0047] 10mmol / L MK2 2μL

[0048] E. coli K-12 cells dipped in a toothpick

[0049] Pfu DNA polymerase 0.5μL

[0050] Sterile double distilled water make up to 50μL

[0051] Pre-denaturation at 94°C for 3 min, then enter PCR cycle: denaturation at 94°C for 0.5 min, annealing at 57°C for 0.5 min, extension at 72°C for 2 min, 4 cycles; then denaturation at 94°C for 0.5 min, annealing at 65°C for 0.5 min, and extension at 72°C for 2 min, 26 Cycles; the final extension at 72°C for 10 min. Take some PCR products for agarose gel electrophoresis dete...

Embodiment 2

[0052] Example 2: Construction of XcmI restriction digestion cassette:

[0053] Primer:

[0054] P1 (forward):

[0055] 5’-GAAGGACCAAGGGTATAATGG CGGCTACAC TA-3’

[0056] P2 (reverse):

[0057] 5’-CTCAAGCTTCCAAGGGTATAATGGACCCCTATTTGTTTAT TTTT-3’

[0058] Template DNA: pET28 (or pET39 and other plasmids containing kanamycin resistance gene or derivative plasmids can also be used).

[0059] PCR reaction system and its parameters:

[0060] 10×Buffer(with Mg 2+ ) 5μL

[0061] 10mmol / L each dNTPs 2μL

[0062] 10mmol / L MK3 2μL

[0063] 10mmol / L MK4 2μL

[0064] Plasmid (500-fold dilution) 2μL

[0065] Pfu DNA polymerase 0.5μL

[0066] Sterile double distilled water make up to 50μL

[0067] Pre-denaturation at 94°C for 3min, enter PCR cycle, denaturation at 94°C for 0.5min, annealing at 45°C for 0.5min, extension at 72°C for 2min, 4 cycles, extension at 72°C for 10min; then denaturation at 94°C for 0.5min, annealing at 65°C for 0.5min, Extension at 72°C for 1 min, 26 cycle...

Embodiment 3

[0068] Example 3: Gene splicing

[0069] Primer:

[0070] MK1:

[0071] 5’-TATGGATCCTGCTCCCCGAACGATTGTGA-3’

[0072] P2 (reverse):

[0073] 5’-CTCAAGCTTCCAAGGGTATAATGGACCCCTATTTGTTTATTTTT-3’

[0074] Template DNA: PCR products obtained in Example 1 and Example 2 (purified and recovered by 3S DNA GelPurification Kit)

[0075] Splicing reaction system and its parameters:

[0076] The products of Example 1 and Example 2 1μL each

[0077] 10mmol / L each dNTPs 4μL

[0078] 10mmol / L MK1 4μL

[0079] 10mmol / L MK4 4μL

[0080] 10×Buffer(with Mg 2+ ) 10μL

[0081] Tag plus DNA polymerase 1μL

[0082] Sterile double distilled water make up to μL

[0083] Pre-denaturation at 94°C for 3min, enter the cycle, denaturation at 94°C for 0.5min, annealing at 58°C for 0.5min, extension at 72°C for 2min, for 30 cycles; final extension at 72°C for 10min. The resulting spliced ​​products were detected by agarose gel electrophoresis, and the remaining products were labeled and stored in a...

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Abstract

The invention provides an expression type pre-T-vector for quickly cloning and expressing the product PCR of the target gene, and a preparation and an application thereof. The advantages of the expression type pre-T-vector mainly lie as follows: the target gene can be cloned and expressed by one step method, thereby omitting complicated recombination processes; the product PCR is not needed to be purified and enzyme cut in construction process, and linearized T-vector can be obtained by the method that the pre-T-vector is single enzyme cut by XcmI with high enzyme cutting efficiency without using other restrictively restriction enzymes; the target gene is not needed to be induced by expensive IPTG, which can ferment engineering bacteria in large scale with lower cost; and the sequencing can be identified after the target gene is expressed, and then transformants whose expression results are up to expectation are sent to the DNA sequencing company to be sequenced, so the sequencing success rate can be improved, and the expenses can be saved.

Description

(1) Technical field [0001] The invention relates to an expression type pre-T vector that can be used for rapid cloning and expression of a PCR product of a target gene, and preparation and application thereof. (2) Background technology [0002] Genetic engineering is to recombine the target gene into an expression vector, and transform the recombinant expression vector into an appropriate host to express the desired protein, thereby exerting benefits. [0003] PCR technology is the most commonly used method to obtain target gene fragments. It is a basic technology in molecular biology and genetic engineering research. It is widely used to amplify known or unknown specific DNA fragments in vitro. There are already T vectors for rapid cloning of PCR products on the market, such as pUCm-T vector. Applicant Lin Chenshui of the present invention and others have applied for a pre-T vector for cloning a target PCR product and a preparation method thereof. Because rapid and effective clo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/66
Inventor 林陈水许明于真真任慧颖杨丹燕
Owner ZHEJIANG UNIV OF TECH
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