Long PCR walking kit and using method and application thereof

A kit and walking technology, which is applied in the field of long PCR walking kits, can solve the problems of cumbersome operation, complicated setting procedures, and many non-specific fragments, and achieve broad application prospects, reduced walking operations, and reduced experimental costs Effect

Inactive Publication Date: 2013-06-19
SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the Tail-PCR setting procedure is complicated. In order to obtain a stable and correct PCR product, it is necessary to repeatedly set the program conditions.
In addition, the use of primers with degenerate base ends and the inherent limitations of the Tail-PCR setup program make this method unable to obtain long unknown flanking fragments and the product has more false positives
Generally speaking, the above methods all have the defects of cumbersome operation, many non-specific fragments, and short fragments (less than 1.5Kb) in one step.
These shortcomings limit its wider application, although obtaining flanking unknown sequences is often faced with work

Method used

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  • Long PCR walking kit and using method and application thereof
  • Long PCR walking kit and using method and application thereof
  • Long PCR walking kit and using method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Long PCR walks were used to amplify unknown DNA sequences flanking the transposon valT of Vibrio alginolyticus E0601-like Vibrio cholerae. Prepare the reagents according to the following formula.

[0031] Kit composition:

[0032] 1) Reagent A: 500 μL of the reaction premix system. Each 10.5 μL contains 1 μL dNTP (dATP, dTTP, dGTP, dCTP each 10 mmol / L), 4 μL MgCl 2 (25mmol / L), 5μL 10×PCR buffer, 0.5μL LA Taq enzyme;

[0033] 2) Reagent B: 10mL ddH 2 O;

[0034] 3) Reagent C: agarose, 6g;

[0035] 4) Reagent D: 1mL of 6×PCR product loading buffer, its components are: 30mmol / L EDTA, 36% (V / V) glycerol, 0.05% (W / V) xylene cyanine, 0.05% (W / V )BPB.

[0036] 5) Reagent E: Goldview nucleic acid dye, 40 μL;

[0037] 6) Reagent F: 10 mL of 50×TAE buffer solution, containing 242 g of Tris, 37.2 g of ETDA, and 57.1 mL of acetic acid per 1 L.

[0038] 7) Primer series, concentration 10 μmol / L, 10 μL each. The sequence is shown as SEQ ID NO: 1-10;

[0039] 8) One primer,...

Embodiment 2

[0052] According to the partial sequence of the rpoB gene of Vibrio vulnificus 1.1758, the nearly full-length gene of rpoB was amplified by long PCR walking. Prepare reagents according to the following formula:

[0053] 1) Reagent A: 300 μL of reaction premix system. Each 11.4 μL reagent A contains 2 μL dNTP (dATP, dTTP, dGTP, dCTP each 10 mmol / L), 4 μL MgCl 2 (25mmol / L), 5μL 10×PCR buffer, 0.4μL LATaq enzyme;

[0054] 2) Reagent B: 20mL ddH 2 O;

[0055] 3) Reagent C: agarose, 10g;

[0056] 4) Reagent D: 1 mL of 6×PCR product loading buffer, its components are: 25mmol / L EDTA, 30% (V / V) glycerol, 0.06% (W / V) xylene cyanide, 0.04% (W / V )BPB;

[0057] 5) Reagent E: Goldview nucleic acid dye, 30 μL;

[0058] 6) Reagent F: 30 mL of 50×TAE buffer solution, containing 242 g of Tris, 37.2 g of ETDA, and 57.1 mL of acetic acid per 1 L;

[0059] 7) Primer series, concentration 15 μmol / L, 10 μL each. The sequence is shown as SEQ ID NO: 1-10;

[0060] 8) One primer, 15 μL, conc...

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Abstract

The invention discloses a long PCR walking kit and a using method and the application thereof. The long PCR walking kit comprises: (1) reagent A, wherein every 9.25-12.5mu L of reagent A contains 1-3 mu L of 10m mol / L dNTP, 3-4mu L of 25m mol / L MgCl2, 5mu L of 10* PCR buffer solution and 0.25-0.5mu L of Taq enzyme; (2) reagent B, 5-20 ml of ddH2O; (3) reagent C, 5-20g of agarose; (4) reagent D, 1-5mL of 6* PCR loading buffer solution; (5) reagent E, 20-100mu L of nucleic acid dye; (6) reagent F, 10-20mL of 50* electrophoretic buffer solution; and (7) 11 primers which are shown in the sequence SEQ ID NO.: 1-11, wherein the content of each primer is 10-50mu L. The long PCR walking kit is low in cost, is obviously simple and rapid compared with other amplification methods, has high efficiency, can obtain a flanking fragment which is longer than 3Kb for one time, is used for acquiring DNA flanking unknown sequence without species restriction, and can be widely used for molecular biological experiments.

Description

technical field [0001] The invention relates to the field of cloning of unknown sequences flanking known DNA fragments, in particular to a long PCR walking kit and its use and application. Background technique [0002] For research related to genetics and molecular biology, obtaining unknown sequences flanking known DNA sequences is an important task that is often faced. For example: analysis of T-DNA insertion sites in plant functional genomics; understanding of bacterial transposon insertion sites and foreign genes carried; obtaining full-length genes based on known partial gene fragments; functional gene promoter analysis, etc. . The traditional approach to obtaining unknown sequences flanking known DNA sequences is to construct libraries and then perform extensive screening of the libraries. This method is not only time-consuming, laborious, and costly, but the final result depends on the quality of the constructed library. Therefore, genome walking based on PCR techn...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P19/34C12R1/63
Inventor 罗鹏苏婷任春华胡超群
Owner SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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