Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

One-step method BbsI enzyme digestion connection fragment assembly method, assembly kit and application

An assembly method and kit technology, applied in biochemical equipment and methods, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of high time and labor costs, and achieve high assembly efficiency, high success rate, and optimized steps Effect

Pending Publication Date: 2022-03-29
上海英基生物科技有限公司
View PDF6 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At the same time, the conventional enzyme digestion ligation construction operation requires enzyme digestion, electrophoresis, and gel recovery of the carrier first, and then similarly amplifies or synthesizes the fragments, and then uses them in the ligation reaction after purification.
However, the enzyme digestion, electrophoresis, gel cutting and recovery steps of the carrier take at least 1 day, and the time and labor costs are relatively high.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • One-step method BbsI enzyme digestion connection fragment assembly method, assembly kit and application
  • One-step method BbsI enzyme digestion connection fragment assembly method, assembly kit and application
  • One-step method BbsI enzyme digestion connection fragment assembly method, assembly kit and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1. Restriction ligation one-step method for encoding sgRNA sequence assembly

[0039] For the sequences of sgRNAs encoding targets of different species, use the one-step enzyme digestion ligation system to assemble the fragments onto the model plasmid, and explore the success probability of the ligation by colony PCR and sequencing. The specific experimental procedure is as follows.

[0040] 1. One pair of random targeting sequences, 2 pairs of mouse Wnt1 targeting sequences designed according to the website, and 3 pairs of targeting plant gRT, OsU6aT, and OsU3T sites in the references. The sequence information is shown in Table 1:

[0041] Table 1. Primers for sgRNA coding sequences of different targets

[0042]

[0043] 2. Configure the annealing system according to Table 2.

[0044] Table 2. Annealing reaction system

[0045]

[0046] Annealing reactions were performed according to Table 3.

[0047] Table 3. Annealing reaction program

[0048]

...

Embodiment 2

[0074] Example 2 The one-step method of enzyme digestion and ligation is used for the assembly of single fragments of different lengths

[0075] For the insertion fragments of different lengths, use the one-step enzyme digestion ligation system to assemble the fragments into approximately model plasmids, and explore the probability of successful connection by colony PCR and sequencing. The specific experimental procedure is as follows.

[0076] 1. Design insert fragments of different lengths, and the sequence information is shown in Table 7.

[0077] Table 7. Primer sequences for amplification of fragments of different lengths

[0078]

[0079] 2. Configure the amplification system according to Table 8.

[0080] Table 8. Amplification system

[0081]

[0082] Set up the amplification program of the model vector according to Table 9.

[0083] Table 9. Amplification Procedure

[0084]

[0085] 3. Purification of amplified fragments

[0086] Prepare a magnetic stan...

Embodiment 3

[0103] Example 3 The one-step method of enzyme digestion and ligation is used for the assembly of different numbers of fragments

[0104] For the insert fragments encoding different lengths, use the one-step enzyme digestion ligation system to assemble the fragments on the approximate model plasmid, and check the success probability of the connection by colony PCR and sequencing. The specific experimental procedure is as follows.

[0105] 1. Design insert fragments of different lengths, and the sequence information is shown in Table 12.

[0106] Table 12 Fragment primer information for 2 or 4 fragment ligation

[0107]

[0108] 2. Configure the amplification system according to Table 8, perform amplification according to the procedures in Table 9, and purify the fragments according to the operation in Example 2.

[0109] 3. According to Table 13, configure the connection system of the model carrier and the fragment.

[0110] Table 13. Multi-fragment linkage system

[01...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention belongs to the technical field of biology, and particularly relates to a one-step method BbsI enzyme digestion connection fragment assembling method, an assembling kit and application. The one-step BbsI enzyme digestion connection assembly kit and the one-step BbsI enzyme digestion connection assembly method are provided for the first time through extensive and deep research and a large number of screening and exploring, BbsI restriction enzyme digestion and T4 DNA polymerase connection are combined into a whole, the steps of clone construction are greatly reduced, the kit and the method can be suitable for assembling short fragments such as encoding sgRNA, and the application prospect is wide. And the operation flow and steps are greatly reduced. Compared with other assembling or constructing kits, the method has the advantages that the assembling efficiency is high, and the method can be used for connecting and assembling multiple fragments with different lengths and is suitable for assembling and constructing multiple modules.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a one-step BbsI restriction fragment assembly method, assembly kit and application. Background technique [0002] With the in-depth research of Crispr technology in this field and the wide application of synthetic biology, the application of type IIS restriction endonucleases in the construction of guide RNA expression systems and the construction of multi-modular clones has increased significantly. The workflow of cloning construction mainly includes: amplification or synthesis and purification of target fragments, and connection of target vectors and fragments. [0003] For common type II restriction endonucleases, the restriction site and the recognition site are at the same position, so that when it is used for cloning construction, the overhanging 4 bases are fixed, which restricts the sequence and cannot be used For multi-fragment connection construction. The Gibs...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12N15/70C12N15/113C12N15/66
CPCC12N15/70C12N15/113C12N15/66C12N2310/20
Inventor 冯延叶胡霞柴智刘绍辉杨敏敏吴典杨佳豪
Owner 上海英基生物科技有限公司
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com