High-molecular-weight T carrier and preparation method thereof

A vector and sequence technology, applied in the field of large molecular weight T vector and its preparation, can solve the problems of high background and low cloning efficiency of non-recombinant transformants, and achieve the effect of improving quality and stability and wide selection range

Inactive Publication Date: 2012-01-04
JIERUI BIOENG SHANGHAI
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The technical problem to be solved by the present invention is that the length of the currently commonly used T vectors is between 2800bp and 3100bp, and for the cloning of foreign fragments with a length between 2500bp and 3500bp, the background of non-recombinant transformants is too high, Insufficiency of lower cloning efficiency, and provide a kind of T vector and preparation method thereof with higher TA cloning efficiency for cloning 2500bp to 3500bp fragments

Method used

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  • High-molecular-weight T carrier and preparation method thereof
  • High-molecular-weight T carrier and preparation method thereof
  • High-molecular-weight T carrier and preparation method thereof

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preparation example Construction

[0030] The invention provides a T vector with higher TA cloning efficiency, especially for cloning fragments that are not easy to distinguish between 2500bp and 3500bp in conventional commercial cloning vectors and a preparation method thereof.

[0031]A T carrier of the present invention comprises: the ColEI ori sequence of pBlue Script II SK (-), the f1 ori sequence of pBlue Script II SK (-), the lacZ gene sequence of pBlue Script II SK (-), pBlue Script II SK (-) ) T3 phage promoter sequence, pBlue Script II SK(-) T7 bacteriophage promoter sequence and a modified multiple cloning site sequence located at both ends, and also includes an exogenous fragment containing amp+ gene. Wherein, the two multiple cloning sites all have 3' protruding T-terminals. In addition to the amp+ gene, the exogenous fragment containing the amp+ gene is artificially extended by adding other sequences, thereby increasing the total length of the entire vector, which is convenient for distinguishing ...

Embodiment 1

[0058] Modification of multiple cloning sites of pBluescriptII SK(-) vector and introduction of XcmI cassette

[0059] For a schematic diagram of plasmid construction, see figure 2 .

[0060] Synthesize MCS_01-MCS_18, totally 18 primers, 18 primers (sequence such as figure 1 shown) were ligated into a target fragment of about 400 bp, ie MCS. Using the vector pENTR3C-dual (Invitrogen) as a template, the ccdb gene was amplified (primers such as figure 1 ccdb_F / ccdb_R as shown). Then, by overlapping PCR, it was fused with the above-mentioned 400bp fragment to obtain the modified XcmI box.

[0061] The PCR reaction system for obtaining MCS is: 5U Pfu, 0.2mmol / LdNTPs, 1×Pfu buffer, 10μmol / L each of primers No. 1 and No. 18, and about 5ng of template (a mixture of primers No. 1-18) in a 50 μL reaction system; The cycle program of PCR amplification is: pre-denaturation at 94°C for 5 minutes, (94°C, 30s; 56°C, 30s; 72°C, 1.5min) × 30 cycles, and last extension at 72°C for 5 mi...

Embodiment 2

[0064] Construct the pre-T vector, use the pJET1.2 (Fermentas) vector containing the AMP+ gene to artificially extend the amp+ gene part of pBluescriptII SK(-), thereby extending the length of the entire vector as a whole

[0065] For a schematic diagram of plasmid construction, see image 3 .

[0066] The plasmid pSKMC that obtains with embodiment 1 construction is template, with XL_F and XL_R primer (see sequence see figure 1 ) for PCR amplification. The reaction system of PCR amplification is: 5U Pfu, 0.2mmol / L dNTPs, 1×Pfu buffer in 50μL reaction system, 10μmol / L of primers, about 5ng of template; the cycle program of PCR amplification is: 94℃ pre-denaturation for 5min , (94°C, 30S; 60°C, 30S; 72°C, 1.5min) x 30 cycles, with a final extension of 5min at 72°C. The PCR products were electrophoresed on a 1% agarose gel and recovered by gel cutting, and the recovered PCR products were digested with BsaI. The pJET1.2 plasmid (Fermentas) was double digested with BpiI and Hi...

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Abstract

The invention discloses a T carrier and a preparation method thereof. A known carrier is a carrier after an improvement of a starting carrier which is pBlueScript II SK(-), pUC18, pUC19, pUC118, pUC119, pUC19, or pGEM serial carriers the polyclone sites of which are replaced with XcmI cases, wherein the XcmI cases comprise ccdB gene sequences and an XcmI restriction enzyme cutting site sequence which is connected to each of two sides of the ccdB gene sequences, and resistance genes in the starting carrier are lengthened by inserting an antisense gene with the length of 1000-2000bp in front and/or back of the antisense gene. The T carrier provided by the invention has higher TA clone efficiency, the TA clone efficiency can reach 100% especially for fragments which are difficult to distinguish for cloning conventional business clone carriers and have the lengths between 2500bp and 3500bp, and the T carrier plays an important role in the filed of genetic engineering.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a large molecular weight T carrier and a preparation method thereof. Background technique [0002] PCR (polymerase chain reaction) is a routine experimental technique in molecular biology. In most cases, it is necessary to clone the PCR amplification product into a plasmid vector for sequencing, in vitro transcription, in vitro translation and other operations on the PCR product. There are many methods for cloning PCR products, but the most direct and convenient method is TA cloning. The so-called TA cloning is to clone the PCR product with a single A tail at the 3' end to a T vector with a single T tail at the 3' end. The theoretical basis for TA cloning is that during PCR amplification, due to the template-independent terminal transferase activity of Taq polymerase, a single deoxynucleotide is added to the 3' end of the PCR product, and four deoxynucleosides are prefe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/66
Inventor 肖爱军周磊赫英俊
Owner JIERUI BIOENG SHANGHAI
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