Novel Gus dyeing liquid and preparation method and application thereof

A preparation method and dyeing solution technology, applied in the field of plant genetic engineering, can solve problems such as defective selection of reagents, and achieve the effects of good dyeing effect, simple preparation process and saving test time.

Inactive Publication Date: 2014-10-01
JIANGSU POLYTECHNIC COLLEGE OF AGRI & FORESTRY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But these two kinds of methods all have its defect, no matter be from the process result of the test or from the selection of reagent all be defective.

Method used

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  • Novel Gus dyeing liquid and preparation method and application thereof
  • Novel Gus dyeing liquid and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0037] Embodiment: Take the preparation of 100 milliliters of x-gluc as an example

[0038] The container used for constant volume in the preparation process is a 100mL volumetric flask. The containers used to preserve the solutions are all 100mL triangular flasks. After all the solutions are prepared, the mouths of the triangular flasks will be sealed with parafilm.

[0039] Step 1: Preparations

[0040] 1. Prepare K 3 [Fe(CN) 6 ] Potassium ferricyanide solution: the required concentration is 50mM;

[0041] Specific method: Weigh 1.647g (gram) and dilute to 100mL sterile deionized water.

[0042] 2. Prepare K 4 [Fe(CN) 6 ] Potassium ferrocyanide: The required concentration is 50mM.

[0043] Specific method: Weigh 2.112g (grams) and dilute to 100mL (milliliters) sterile deionized water.

[0044] 3. Prepare PBS phosphate buffer:

[0045] First prepare liquid A: the composition of liquid A is NaH 2 PO 4 2H 2 O Sodium dihydrogen phosphate dihydrate, weigh 3.12g (grams...

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Abstract

The invention discloses a novel Gus dyeing liquid preparation method which comprises the following steps: (1) preparing a K3[Fe(CN)6] solution and a K4[Fe(CN)6] solution; (2) preparing a PBS (phosphate buffer solution); and (3) preparing dyeing liquid, namely dissolving 5-bromine-4-chlorine-3-benzpyrole-beta-glucuronide ester in methyl alcohol, and sequentially adding the K3[Fe(CN)6] solution, the K4[Fe(CN)6] solution, methyl alcohol, 10% Triton-100 and the PBS after 5-bromine-4-chlorine-3-benzpyrole-beta-glucuronide ester is completely dissolved. The novel Gus dyeing liquid preparation method is relatively simple in preparation process and easy to operate; all the reagents are non-toxic and harmless reagents and cannot injure a human body or pollute an environment; the prepared dyeing liquid has a good dyeing effect; generally, the dyeing liquid can develop a color after 1 hour compared with other dyeing liquid needing a night, so that the test time is shortened.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and in particular relates to a new Gus staining liquid used in transgenic plants and its preparation method and application. Background technique [0002] The gus gene (β-glucuronidase gene) is currently the most widely used reporter gene in transgenic plants. β-glucuronidase is a hydrolase that uses β-glucuronide esters as substrates, and its reaction products can be detected by various methods. Most plants have no background activity of glucuronidase, so the gus gene is widely used as a reporter gene in plant genetic engineering. Taking X-Gluc (5-bromo-4-chloro-3-indole-β-glucuronide) as the reaction substrate, soak the test material in the buffer solution containing the substrate. If the tissue cells are transfected with the gus gene and express Gus, under suitable conditions, the enzyme can hydrolyze X-Gluc to produce a blue product. This is an indigo dye formed by oxidat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N1/30
Inventor 王媛花颜志明
Owner JIANGSU POLYTECHNIC COLLEGE OF AGRI & FORESTRY
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