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Vector for non-background directed cloning of PCR products, preparation method thereof and application thereof

A technology without background and carrier, applied in the field of genetic engineering, can solve the problems of increasing the experimental cost, increasing the workload, and the cloning positive rate cannot reach 100%, and achieves the steps of reducing the experimental cost, low cost, and simplifying the cloning operation and screening. Effect

Inactive Publication Date: 2012-08-08
WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Treatments such as carrier dephosphorylation can reduce the probability of carrier self-ligation, but the PCR primers need to be phosphorylated, which greatly increases the cost of the experiment. Due to the problem of carrier dephosphorylation efficiency, the positive rate of clones is far from reaching 100%. %, PCR product blunt-end clone insertion positive and negative screening also increases the workload for large-scale cloning
[0005] In summary, although the direct cloning of PCR products has the advantages of simple operation in large-scale cloning construction, it still needs to be optimized to a large extent in cloning screening.

Method used

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  • Vector for non-background directed cloning of PCR products, preparation method thereof and application thereof
  • Vector for non-background directed cloning of PCR products, preparation method thereof and application thereof

Examples

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Effect test

example 1

[0043] Intermediate vector pBP1 construction:

[0044] 1) Using specific primers (forward primer: SEQ ID No: 2, reverse primer: SEQ ID No: 3) with pUC18 vector (purchased by TAKARA company) as a template with high-fidelity DNA polymerase KOD-Plus-Neo (TOYOBO Purchased by the company) to amplify the vector framework including Amp resistance gene, replicon, and lac promoter elements (amplification system: 10×PCR Buffer for KOD-Plus-Neo 5μl, 2mM dNTPs 5μl, 25mM MgSO 4 3 μl, 10 μM forward primer 1 μl, 10 μM reverse primer 1 μl, pUC18 DNA 100ng, ultrapure water was added to the final system to 50 μl, and finally KOD-Plus-Neo 1U was added), and a commercial PCR recovery kit (Beijing Biotech Biotechnology Co., Ltd.) to obtain the vector framework fragment after purification, and then digest the vector framework fragment with endonucleases BglII (purchased by TAKARA Company) and KpnI (purchased by TAKARA Company) that recognize the restriction sites at both ends of the fragment, and ...

example 2

[0048] Construction of a vector pDNB1 for directional cloning of PCR products without background:

[0049] 1) Design and synthesize an XcmI box of about 800bp, which contains PstI recognition site, XcmI recognition site, lac promoter, ccdB negative selection marker gene, XcmI recognition site, SphI recognition site and other characteristics. The sequence is shown in the sequence list SEQ ID No: 1;

[0050] 2) Digest the XcmI box with PstI (purchased by TAKARA Company) and SphI (purchased by TAKARA Company), and use a commercial DNA gel recovery kit (Beijing Biotech Biotechnology Co., Ltd.) to recover the digested fragments;

[0051] 3) Digest the intermediate vector pBP1 with PstI (purchased by TAKARA Company) and SphI (purchased by TAKARA Company), and use a commercial DNA gel recovery kit (Beijing Biotech Biotechnology Co., Ltd.) to recover the digested fragments;

[0052] 4) Mix the XcmI box recovered by enzyme digestion and the intermediate carrier pBP1 recovered by enzym...

example 3

[0054] Application of a background-free vector for directional cloning of PCR products in TA ligated cloning:

[0055] 1) Treat the pDNB1 plasmid with restriction endonuclease XcmI (purchased by New England Biolabs), and after 3 hours, electrophoresis and recover to obtain a linear plasmid vector of about 2.5kb, which is named pDNB-T, which can be directly used for TA cloning of PCR fragments T carrier;

[0056] 2) Using specific primers (forward primer: SEQ ID No: 6, reverse primer: SEQ ID No: 7) to use the genome of Escherichia coli K12 (gifted by Professor Qi Qingsheng of Shandong University) as a template, use Taq enzyme (from Beijing Purchased by Dingguo Biotechnology Co., Ltd.) to amplify SD-lacZa-1 fragment of about 280bp (amplification system: 5 μl of 10×Taq Buffer, 5 μl of 2mM dNTPs, 1 μl of 10 μM forward primer, 1 μl of 10 μM reverse primer, large intestine Bacillus K12 genome 100ng, ultrapure water was added to the final system (50 μl, and Taq enzyme 1U was added a...

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Abstract

The invention discloses a vector for non-background directed cloning of PCR products, a preparation method thereof and an application thereof. The vector is characterized in that an XcmI box is introduced before the vector screens and labels gene, and the XcmI box is characterized in that a protruded dT end is formed after XcmI enzyme digestion, an inactive ribosome binding site is formed before gene screening and labeling and after sequence splicing, and the screened and labeled gene cannot express during self-joining of the vector. The protruded dT end formed after the XcmI enzyme digestionof the vector prepared in the invention can be applied to TA cloning, and can be filled in by a T4DNA polymerase to form blunt ends, so as to be applied to blunt end joining; the method of the invention has the advantages of easy implementation, and low cost; and effects of no background and directed cloning are reached in the application of two cloning methods.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a carrier capable of directional cloning of PCR products without background, a method for preparing a carrier capable of directional cloning of PCR products without background, and at the same time, a carrier capable of directional cloning of PCR products without background carrier application. Background technique [0002] PCR (polymerase chain reaction) is a routine experimental technique in molecular biology. Most of the gene function research requires the target fragment to be amplified by PCR and then cloned into the corresponding vector for research. At present, there are many methods for cloning PCR products, including the traditional enzyme-cut ligation technology, the newly developed Gateway that does not rely on enzyme-cut system, Topo cloning independent of T4 DNA ligase And a ligase-independent cloning system (ligation-independent cloning, L...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/64
Inventor 胡杨波冯立鹏陈士云
Owner WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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