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Method for preparing nucleic acid molecular weight label

A molecular weight and nucleic acid technology, applied in the field of molecular biology, can solve problems such as low production efficiency and cumbersome steps of nucleic acid molecular weight marking

Inactive Publication Date: 2008-05-14
TONGJI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] Aiming at the shortcomings of cumbersome steps and low production efficiency in the production of nucleic acid molecular weight markers in the prior art

Method used

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  • Method for preparing nucleic acid molecular weight label
  • Method for preparing nucleic acid molecular weight label
  • Method for preparing nucleic acid molecular weight label

Examples

Experimental program
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Effect test

Embodiment 1

[0033] figure 1 It is the multiple cloning restriction site of the psp72 plasmid. In this experiment, two restriction sites, Sal I and BamHI, were selected, and the vector after digestion was recovered for 1.5 hours to obtain the double digestion vector.

[0034] The synthesized sequence is a single-stranded oligonucleotide, which is synthesized by the company according to the designed sequence (Shanghai Sangon Bioengineering Technology Service Co., Ltd., Lot No: AA26474, AA26475). The two strands are complementary. Anneal to form double strands first. Annealing conditions are as follows:

[0035] Nuclease-free H 2 O 40ul

[0036] 5×annealing buffer 20ul

[0037] DNA oligoA 20ul

[0038] DNA oligoB 20ul

[0039]

[0040] 100ul

[0041] 95°C×2min, drop 0.5°C every 40 seconds, and store at 4°C after dropping to 25°C (annealing buffer is a product of Biyuntian Company)

[0042] as figure 2 As shown, the bold it...

Embodiment 2

[0050] Figure 4 As shown, inserts of different lengths are used to make markers, and several bases can be randomly added to the original insert. In this embodiment, 50 bases (lowercase letters) are added on the original basis to make the primers The length of the sequence between them reaches 150bp, while the original enzyme cutting site and primer binding site remain unchanged. It can be seen that markers of various molecular weights can be produced by designing and synthesizing insert sequences of different lengths, saving time and effort.

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Abstract

The invention discloses a method for preparing nucleic acid molecular weight standard through one-time polymerase chain reaction (PCR) to amplify all bands required by a complete marker. Two synthetical single-chain nucleotides are annealed, when double chains are formed, the two synthetical single-chain nucleotides are connected on carriers by the same tail end sequence. After conventional procedures such as transformation, etc., ascherichia coli clone is produced. Under the conditions of existing primers, dNTP, DNA polymerase and buffer fluid and according to a certain circulation conditions, the PCR amplification is carried out. The circulation amount and an amplification system are adjustable according to different requirements; the obtained material is added with appropriate amount of bromophenol blue, and the finished products are prepared. During the next production, the conservation bacteria can be cracked and used as a template (or directly adopting plasmid) to do the PCR amplification again, and the products can be fast and effectively prepared abundantly, which saves manpower, material and financial resources.

Description

technical field [0001] The invention belongs to the field of molecular biology and relates to the preparation of nucleic acid molecular weight markers. Background technique [0002] At present, the production of nucleic acid molecular weight markers at home and abroad uses the method of enzymatic digestion. Different endonucleases are selected according to the size of the required marker and different plasmids are cut, and then the digested products are mixed according to a certain ratio and then added with an appropriate amount of loading buffer. into products. However, as the market demand increases, it is necessary to repeat this step many times to prepare enough products to meet the needs of production and consumption. Increased production cost and labor time. Contents of the invention [0003] Aiming at the shortcomings of cumbersome steps and low production efficiency in the production of nucleic acid molecular weight markers in the prior art. The main purpose of ...

Claims

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Application Information

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IPC IPC(8): C12P19/34C12N15/11C12N15/65C12N15/10
Inventor 陆海瑛张军
Owner TONGJI UNIV
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