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Preparation method of high-yield nucleic acid fragments

A nucleic acid fragment, high-yield technology, applied in the biological field, can solve problems such as low economic efficiency, increased material demand, and extended production cycle

Pending Publication Date: 2021-06-08
JIANGSU YAOHAI BIOLOGICAL PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Usually the recombinant plasmid contains only one copy of the target nucleic acid fragment. When the demand for the target nucleic acid fragment is relatively low, the low-yield target nucleic acid fragment can also meet the demand, but when the demand is relatively large, the yield of the target nucleic acid fragment is low. It becomes extremely prominent, the demand for materials increases, and the production cycle is prolonged, which directly leads to low economic benefits.

Method used

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  • Preparation method of high-yield nucleic acid fragments

Examples

Experimental program
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Effect test

Embodiment 1

[0035] The construction of embodiment 1 recombinant plasmid

[0036] S1. Take the pUC57-Kan-Yh plasmid strain containing the target gene, inoculate it in LB liquid medium containing 50 μg / mL Kan, shake it at 37°C and 220 rpm overnight, take the overnight culture, and use the plasmid to extract a small amount The kit performs plasmid extraction to obtain the pUC57-Kan-Yh target plasmid.

[0037] S2. Take 10 μg of the pUC57-Kan-Yh target plasmid, digest it with the restriction endonuclease KpnI, and digest it at 37°C for 40 minutes. After the reaction solution is separated by 1% agarose gel electrophoresis, the target of about 3759 bp is recovered by cutting the gel. Fragment bands and 2579bp vector fragments, and then purified by DNA gel recovery kit to obtain Yh target gene fragments and vector backbone fragments, such as figure 1 shown.

[0038] S3. Add calf intestinal alkaline phosphatase (CIAP) to the recovered carrier skeleton fragment solution for dephosphorylation trea...

Embodiment 2

[0048] Example 2 Screening of high-yielding plasmid strains and analysis of the target gene fragment yield of recombinant plasmids:

[0049] 1. Screening of high-yielding plasmid strains

[0050] The 5 newly-made plasmid strains and original plasmid strains that were correctly identified were inoculated in 50 μg / mL Kan-resistant LB liquid medium, and cultured overnight at 37°C and 220 rpm with shaking. Measure the OD of each bacterial solution at 600nm with a spectrophotometer 600 value. Adjust each bacterial solution to an appropriate OD with sterile water 600 , take an appropriate volume of bacterial liquid, extract the plasmid with a plasmid mini-extraction kit, and use the same volume (100 μL) for elution. Then measure the concentration of each plasmid solution with an ultra-micro spectrophotometer, and calculate the content of the plasmid and the content of the target gene fragment in each sample. According to the content of the target gene segment, the plasmid with h...

Embodiment 3

[0054] The separation and purification analysis of the target gene fragment of embodiment 3

[0055] (1) Plasmid amplification and extraction

[0056] The above-mentioned recombinant plasmid strains and original plasmid strains were respectively inoculated in Kan-resistant LB liquid medium containing 50 μg / mL, and cultured overnight at 37°C and 220 rpm with shaking. Bacteria were collected by centrifugation at 8000×g for 2 min to collect the bacteria, and the plasmids were extracted respectively.

[0057] (2) Enzymatic digestion

[0058] Take 1 mg of the recombinant plasmid and the original plasmid and digest them with the restriction endonuclease KpnI, and detect them by electrophoresis until the digestion is complete. Figure 7 shown.

[0059] (3) Purification of target gene fragments

[0060] Take a certain amount of digestion solution and use AKTA for sample loading analysis. Separation and purification by chromatographic column NanoQ-15L, the target peak and carrier ...

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Abstract

The invention is applicable to the technical field of biology, and provides a preparation method of high-yield nucleic acid fragments. The method comprises the following steps of introducing recognition sites of the same restriction enzyme at the 5' and 3' ends of a target gene sequence so as to obtain target gene fragments through single enzyme digestion, and ensuring the uniformity of the fragments; connecting the target gene fragments and a vector skeleton into a recombinant plasmid containing the target gene fragments of at least 2Copy in enzyme digestion and connection modes; amplifying and collecting the recombinant plasmid, and carrying out enzyme digestion on the recombinant plasmid by utilizing single restriction enzyme to obtain enzyme digestion liquid; and separating and purifying the enzyme digestion liquid by using AKTA through anion exchange resin to obtain the target gene fragments. According to the method, the high yield of a single batch of target gene fragments is realized, the scale and batch quantity of fermentation and purification are reduced, the production period is shortened, the equipment utilization rate is increased, and the economic benefit is increased.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a method for preparing high-yield nucleic acid fragments. Background technique [0002] The preparation of nucleic acid fragments is mainly obtained by synthesis, PCR amplification method and enzyme digestion after plasmid amplification. The PCR method is difficult to ensure the uniformity between batches due to the existence of low-probability mutations in the process of in vitro polymerase recognition, addition, and cutting. , and nucleic acid fragments obtained from plasmids can effectively ensure the stability of gene sequences due to the complex replication mechanism in cells, and restriction endonucleases are specific cuts to ensure the batch-to-batch consistency of target nucleic acid fragments. Therefore, it is generally more inclined to use plasmid digestion to obtain a large number of target nucleic acid fragments with uniform quality. [0003] In the preparation process o...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12N15/63
CPCC12N15/10C12N15/63
Inventor 张永正马诗敏张征立李洋康涛
Owner JIANGSU YAOHAI BIOLOGICAL PHARM CO LTD
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