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Chlamydomonas reinhardtii efficient library building method using square wave electroporation

A technology of Chlamydomonas reinhardtii and square wave, which is applied in the field of efficient library construction of Chlamydomonas reinhardtii by using square wave electric shock, to solve the problem of exogenous DNA transformation, good promotion and application value, and small genome effect

Active Publication Date: 2016-05-04
XUZHOU NORMAL UNIVERSITY
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Problems solved by technology

[0006] The purpose of the present invention is to provide a method for using square wave electric shock to integrate exogenous DNA into the chromosome DNA of Chlamydomonas reinhardtii, especially a method for efficiently building a library of Chlamydomonas reinhardtii by using square wave electric shock, so as to solve the problem of Chlamydomonas DNA transformation efficiency problem

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  • Chlamydomonas reinhardtii efficient library building method using square wave electroporation
  • Chlamydomonas reinhardtii efficient library building method using square wave electroporation

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Embodiment Construction

[0024] Such as figure 1 and figure 2 As shown, the present invention includes the following steps: 1. Prepare cells, plates and prepare DNA: Ultra-clean workbench ultraviolet sterilization for 15 minutes in advance, inoculate fresh Chlamydomonas 21gr cells into sterile TAP liquid blowing bottles, at 23 ± 0.5 °C, 8000Lx light intensity, continuous light and aeration culture, 3-4 days, until the cell solubility is 1-2×10 7 cells / ml, transfer the cells again to a fresh sterile TAP liquid insufflation bottle, and dilute to an initial concentration of 1×10 6cells / ml, continue to culture under continuous light for 20 h, and make the final concentration reach 4×10 6 cells / ml can be used for electroshock transformation. The TAP liquid medium plate includes: TAP salt solution 25ml / L, phosphate solution 0.375ml / L, Hutner trace element 1ml / L, acetic acid 1ml / L, Tris2.42g / L, 121 ℃ high pressure steam sterilization 20min ; The TAP salt solution is: NH 4 Cl15g / L, MgSO 4 ·7H 2 O4g / L...

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Abstract

The invention discloses a chlamydomonas reinhardtii efficient library building method using square wave electroporation. The method includes the following steps that chlamydomonas reinhardtii is continuously illuminated at 23+ / -0.5 DEG C in a TAP fluid medium, subjected to aerobic culture at 8000 Lx light intensity and then cultured and treated after being transferred, resistance DNA fragments are transformed through square wave electroporation, resistance DNA is randomly integrated into chlamydomonas reinhardtii chromosomes, a resistance screening plate is coated with a product obtained after overnight low light recovery, and a mutant library causing random insertion mutation is obtained after culture of a light period lasting 7 days. The method has the advantage that a large number of randomly-inserted transformants can be efficiently obtained to build the chlamydomonas reinhardtii mutant library.

Description

technical field [0001] The present invention relates to a method for using square wave electric shock to integrate exogenous DNA into chromosome DNA of Chlamydomonas reinhardtii, in particular to a method for using square wave electric shock to randomly insert resistant DNA into Chlamydomonas reinhardtii chromosome DNA to construct randomly inserted Chlamydomonas reinhardtii Methods for mutant libraries. Background technique [0002] Chlamydomonas reinhardtii ( Chlamydomonas reinhardtii ) is a eukaryotic single-celled green algae, because of its fast growth, easy cultivation, small and annotated genome, relatively mature biotechnology methods, and the molecular mechanism of intracellular physiological processes is highly similar to that of higher animals and plants, so it is currently a plant One of the model organisms common to animals. Using forward genetic insertion mutagenesis to study the relationship between phenotype and gene, and to further reveal the biological fu...

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Application Information

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IPC IPC(8): C12N15/90C12N1/13
CPCC12N1/12C12N15/90
Inventor 王亮范志月杨丽婧杨丰源陆军郑元林
Owner XUZHOU NORMAL UNIVERSITY
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