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Plasmid transforming method

A plasmid and fragment technology, applied in the fields of molecular biology and genetic engineering, can solve the problem of low plasmid transformation efficiency, and achieve the effect of improving transformation efficiency and high transformation efficiency.

Active Publication Date: 2018-09-07
LANZHOU UNIVERSITY OF TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Using this method for plasmid transformation has a high transformation rate, which can effectively solve the problem of low plasmid transformation efficiency in practical operations.

Method used

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] 1. PCR amplification of the exogenous linear targeting fragment containing the kana resistance gene for knocking out the ygiZ gene:

[0041] (1) Using the plasmid pKD4 containing the Kanak resistance gene as a template, use primers K-ygiZ-F and K-ygiZ-R to amplify the Kanak resistance gene linear targeting fragment "homologous arm + FRT + Kan Sequence+FRT+homology arm", after the amplification is completed, it is detected by 1% agarose gel electrophoresis.

[0042] The amplification system is shown in Table 1.

[0043] Table 1 PCR system of exogenous kana resistance gene linear targeting fragment

[0044] Reagent name

volume

5×prime STAR buffer (Mg 2+ +plus)

10 μL

dNTP mixture (2.5mM each)

4μL

K-ygiZ-F

1μL

K-ygiZ-R

1μL

template

1μL

Primer STAR HS DNA polymerase (2.5units / μL)

0.5μL

Sterilized distilled water

Up to 50μL

[0045] (2) PCR amplification conditions: pre-denatura...

Embodiment 2

[0071] (1) Loss of the recombinant strain E.coli DH5α::Kan::△ygiZ Kanna resistance fragment (also can be understood as: deletion or knockout)

[0072] The temperature-sensitive plasmid pCP20, which can encode the recombinant protein FLP, was introduced into the correctly identified E.coliDH5α::Kan::△ygiZ recombinant strain by electroporation, revived and cultured at 30°C and 150r / min for 1 hour, and 200 μL was spread on the containing 35 μg / mL chloramphenicol on LB plate medium, cultured at 30°C for 8 hours, then increased to 42°C for overnight culture, heat-induced expression of FLP recombinase, and plasmid loss.

[0073] (2) Screening and identification of mutant strain E.coli DH5α::△ygiZ

[0074] Use a sterile toothpick to randomly pick 5 single colonies grown on the chloramphenicol plate, and inoculate each single colony on the chloramphenicol plate, the ampicillin plate and the plate without antibiotics, and culture at 37°C for 12-16 hours. The colony that can only grow ...

Embodiment 3

[0083] Determination of Transformation Efficiency of Mutant Strain E.coli DH5α::△ygiZ

[0084] with 100mM CaCl 2 Prepare wild-type E.coli DH5α and mutant strain E.coli DH5α::△ygiZ competent cells respectively, and dilute plasmids pUC19, pET-32a, p1304 to 5ng / μL. Take 2 μL and add them to 100 μL of the two competent states, mix gently, and ice-bath for 30 minutes. Heat shock at 42°C for 90s, ice-bath for 2min, then add 900μL LB culture medium and re-incubate at 37°C for 50min at 180r / min. Take 100 μL and spread on LB plate respectively, culture overnight at 37°C and count.

[0085] Transformation efficiency = (number of plate colonies × dilution factor) / plasmid mass (ng)

[0086] The result is as Figure 4-Figure 7 As shown, the results show that the transformation efficiencies of the competent cells of wild-type E.coli DH5α and mutant E.coli DH5α::ΔygiZ to plasmid pUC19 are 2.58×10 6 CFU / μg and 5.28×10 6 CFU / μg;

[0087] The transformation efficiency of plasmid pET-32...

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Abstract

The invention discloses a plasmid transforming method and relates to the fields of molecular biology and genetic engineering. The plasmid transforming method comprises the steps as follows: target exogenous plasmid and a competence host cell with an ygiZ gene knocked out are mixed. The ygiZ gene of the host cell is knocked out, so that the transforming rate is increased. The method has the characteristic of high transforming efficiency for large plasmid. Compared with wild type host cells, the host cell with the ygiZ gene knocked out has the transforming efficiency improved by 2.05 times for plasmid pUC19, 2.60 times for pET-32a and 1.67 times for plasmid p1304. The method greatly improves the plasmid transforming efficiency and has very great significance in the molecular biology field and the genetic engineering field.

Description

technical field [0001] The invention belongs to the fields of molecular biology and genetic engineering, and specifically relates to a method for transforming plasmids. Background technique [0002] CaCl 2 The preparation of Escherichia coli (Escherichia.coli) competent cells is one of the most basic and commonly used operating techniques in molecular biology experiments, and is widely used in the research of recombinant plasmid transformation, gene cloning and gene library construction. Although theoretically CaCl 2 The most ideal transformation efficiency of competent cells prepared by the method can reach 5×10 6 ~2×10 7 cfu / μg DNA, however, in practice the conversion rate is often lower than 10 5 , sometimes as low as 10 3 , unable to meet the needs of relevant experimental research. Contents of the invention [0003] In view of the problems existing in the above-mentioned prior art, the object of the present invention is to provide a method for transforming plasm...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70
CPCC12N15/70
Inventor 王永刚杨光瑞李渊利陈吉祥马建忠
Owner LANZHOU UNIVERSITY OF TECHNOLOGY
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