100bp gradient ribonucleic acid molecular weight marker and its preparation

A molecular weight and marker technology, used in the introduction of foreign genetic material, DNA/RNA fragments, recombinant DNA technology, etc. using vectors

Inactive Publication Date: 2005-08-17
JIANGSU INST OF PARASITIC DISEASES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

After reviewing the domestic patent literature, no reports have been found on the design of 11 pairs of specific primers using the plasmid PBR322 as a template, and the preparation of 100bp gradient DNA molecular weight markers composed of 11 DNA bands by polymerase chain reaction.

Method used

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  • 100bp gradient ribonucleic acid molecular weight marker and its preparation
  • 100bp gradient ribonucleic acid molecular weight marker and its preparation
  • 100bp gradient ribonucleic acid molecular weight marker and its preparation

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Experimental program
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Effect test

Embodiment 1

[0168] The preparation method of the 100bp gradient DNA molecular weight marker specifically comprises the following steps.

[0169] 1 Preparation of plasmid pBR322DNA

[0170] The bacteria containing the plasmid pBR322 were inoculated into 3 ml of LB broth medium (containing 100 μg / ml of ampicillin), and cultured overnight at 37° C. with shaking. Bacteria were collected by centrifugation at 12000g, and then the plasmid DNA purification kit was used to purify the pBR322 plasmid. The process of plasmid DNA extraction was carried out according to the instructions of the kit.

[0171] 2. Preparation of DNA bands - polymerase chain reaction

[0172] Take 11 0.25ml PCR thin-walled centrifuge tubes and mark them with 100bp, 200bp, 300bp, 400bp, 500bp, 600bp, 700bp, 800bp, 900bp, 1000bp, 1200bp, and add Various reagents for polymerase chain reaction:

[0173] 10×Taq DNA polymerase buffer 10μl

[0174] 4×dNTP 8μl

[0175] Forward primer 2μl

[0176] Reverse primer 2μl

[0177] ...

Embodiment 2

[0193] Embodiment 2: for identifying positive clonal colonies

[0194] Pick colony 1 and colony 2 on the transformation plate with an inoculation loop, suspend in deionized water, and boil to lyse. Using this cleavage as a template, use gene-specific primers for PCR amplification, take 5 μl of the molecular weight marker of the present invention and 10 μl of the amplification products of colony 1 and colony 2, and perform agarose gel electrophoresis. Observe the results below. The results showed that the amplified product of colony 1 produced a single DNA band with a molecular weight of 700-800bp, which was similar to the size of the cloned target gene, which preliminarily proved that the colony was a positive clone colony, while the amplified product of colony 2 did not have any DNA Bands appear, proving that the colony is a negative clone. Such as image 3 .

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Abstract

The 100 bp gradient DNA molecular weight marker and its preparation belongs to the field of molecular biology reagent preparing technology. The molecular weight marker is one kind of mixture comprising 11 DNA stripes with the lengths of 100 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp,1000 bp, and 1200 bp separately and the concentration of 4 ng / microliter. The preparation process includes simultaneous synthesis of the 11 DNA stripes through PCR with 11 pairs of specific primers and plasmid PBR322 as template DNA in the presence of Taq DNA polymerase, Taq DNA polymerase buffering liquid and 4 kinds of ribotides (dTTP, dATP, dCTP and dGTP), in the same amplification condition in a PCR instrument; and the subsequent purification and mixing. The present invention features the fast, precise and mass preparation of DNA molecular weight markers of different lengths in the set target.

Description

technical field [0001] The invention uses polymerase chain reaction (PCR) technology to prepare DNA molecular weight markers with a gradient of 100 bp, and belongs to the field of molecular biology reagent preparation. Background technique [0002] In the process of DNA synthesis and various methods of genetic manipulation, it is necessary to monitor the size change of the synthesized or manipulated DNA molecule. The common method is to combine the DNA molecule to be tested with a DNA mixed standard substance of known molecular weight simultaneously Perform electrophoresis, and judge the size of the DNA molecule to be tested by comparing the migration degree of the DNA molecule to be tested with the DNA molecule standard substance during electrophoresis. Therefore, it is necessary to prepare some standard DNA of known molecular weight. There are several ways to prepare standard DNA: [0003] chemical synthesis [0004] Combine four different deoxyribonucleotides: adenine ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/65
Inventor 余传信殷旭仁
Owner JIANGSU INST OF PARASITIC DISEASES
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