Plasmid extraction can be divided into three parts

1 lyse cells

Separation of plasmid DNA and chromosome DNA

3 purification

Here is a paper:

Construction and identification of eukaryotic expression plasmid of mouse CD40Ig gene

1 materials and methods

1. 1 experimental animal Balb-c mice, 6 weeks old, female, were purchased from the Laboratory Animal Department of Peking University Medical College.

1.2 plasmid, strain, cell strain and main reagent plasmid pEGFP-N 1, pGEM-T vector plasmid, Escherichia coli. DH5A (Tiangen Company). Low generation HEK293 cell line (original Zhengyang). Admax (TM) Kit E (Microbix Biosystems, Inc.). Taq enzyme, T4 DNA ligase (Takara Company). XmaⅰI, Bgl, Hindⅲ(Biolabs) in restriction endonucleases. Trizol reagent, Lipofectamine 2000(Invitrogen). Reverse transcription kit, PCR Master Mix(MBI). Plasmid Extraction and Gel Purification Kit (QIAGEN). DMEM high glucose medium, fetal bovine serum, pancreatin (Gibco). Primer synthesis was completed by Shanghai Gong Sheng Company, and gene sequencing was completed by Beijing Nuosai Gene Research Center Co., Ltd. ..

1.3 Construction of eukaryotic expression plasmid of mouse CD40Ig gene

1.3. 1 Extraction of total RNA from mouse spleen: The mice were killed by dislocation of cervical vertebra and the spleen was taken out. After grinding with liquid nitrogen, total RNA was extracted with Trizol reagent. The method is carried out according to the product specification.

Preparation of CD40, IgG2a Fc and GFP genes of 1.3.2: cDNA was synthesized by RT-PCR with mouse spleen total RNA as template and oligo-dT as primer, and then amplified by PCR with CD40 and mIgG Fc specific primers as template and pEGFP-N 1 plasmid as template. Primers and PCR reaction conditions are shown in Table 650.

1.3.3 construction of plasmid pGEM-IgG: the PCR products of IgG Fc and pGEM-T plasmid were subjected to TA ligation reaction, that is, 10μl PCR products were added with 2μl pGEM-T vector and 10 U T4 ligase, and the temperature was 16℃ overnight. 2μl of ligation product was transformed into Escherichia coli DH5α by calcium chloride method, and white spot clones containing ampicillin resistance were screened by blue white spot method. Plasmid pGEM-IgG was extracted and sequenced overnight in 3 ml LB medium (containing ampicillin 100 μg/ml) at 37℃ and 80 r/min.

Construction of 1.3.4 plasmid p5 16-IgG: pGEM-IgG and pDC5 16 were digested with Bgl II respectively, and the products were purified by 1.0% agarose gel electrophoresis. IgG fragment and pDC5 16 linear vector were ligated overnight with T4 ligase 16 at the molar concentration ratio of 7∶ 1. Take 5μl of the ligation product to transform the competent Escherichia coli DH5α, screen the forward inserted recombinant with the combination of pDC5 16-f and mIgG-r primers (see Table 2), then culture the PCR positive recombinant, extract the plasmid and sequence it.

Construction of 1.3.5 plasmid p5 16-CD40-IgG fusion gene: The PCR products of CD40 and p5 16-IgG were digested with Xma I respectively, and the CD40 fragment was ligated with pDC5 16-IgG linear vector (method as above). After transforming E.coli DH5α, colony PCR screening was carried out with the combination of pDC5 16-f and CD40-r primers (see Table 2), and the positive recombinant was cultured, and the plasmid was extracted and sequenced.

1.3.6 PDC 5 16-CD40-IgG-GFP plasmid construction: The PCR products of GFP and PDC 516-CD40-IgG were digested with Hind enzyme respectively, and the GFP fragment was connected with pDC5 16-CD-IgG linear vector (ibid.

Batch preparation of1.3.7 PDC 516-CD40-IgG-GFP plasmid: according to the instructions of Qiagen endo free plasmid purification kit, the product was confirmed by 1% agarose gel electrophoresis (see Figure 2), the OD value was measured by ultraviolet spectrophotometer, filtered and sterilized, and stored at -20℃ for later use.

1.3.8 Transfection of 293 cells with recombinant plasmid: 2.0× 105 cells were inoculated into 6-well plate 2 ml DMEM containing antibiotic-free serum, and cultured in an incubator containing 5% CO2 at 37℃ for 24 h, so that the cells covered 60% ~ 70% of the plates during transfection. The recombinant plasmid was transfected with Lipofectamine 2000 transfection reagent, and two solutions A and B were prepared in a sterile EP tube. Solution A: Dissolve 4.0μg DNA in 250μl serum-free DMEM and mix gently; Solution B: Dissolve 10μl Lipofectamine 2 000 in 250μl serum-free medium, gently mix and incubate at room temperature for 5 min. Solution A and solution B were mixed and incubated at room temperature for 20 minutes to form a liposome /DNA complex. Replace the old culture medium in 6-well plate, add 2 ml DMEM containing serum and without antibiotics again, and add 500μl liposome /DNA complex drop by drop. Incubate in an incubator containing 5% CO2 and 37℃ for 24 h, and then change the liquid.

Two results

2. 1 The extracellular domain of mouse CD40, IgG2a Fc fragment and GFP gene primers CD40-f and CD40-r were obtained, and the gene fragment of mouse CD40 extracellular domain was 572 bp. The fc fragment of mouse igg2a amplified by primers mIgG-f and mIgG-r is 700 bp. The GFP gene fragment amplified by primers GFP-f and GFP-r is 765 bp. The result of agarose gel electrophoresis is shown in figure 1.

2.2 Successful construction of eukaryotic expression plasmid PDC 5 16-CD40-IgG-GFP

2.2. construction of1PDC 516-IgG: the pGEM-T vector is 3 000 bp, and the constructed pGEM-IgG plasmid is 3 700 bp. PGEM-T plasmid contains T7 primer sequence at 1 10 bp upstream of multiple cloning site. T7+mIgG-r primer combination can amplify a fragment of about 8 10 bp, and T7 primer sequencing confirmed the correctness of IgG sequence. The IgG fragment was connected with the linear vector pDC5 16, and the correctness of the IgG insertion sequence was confirmed by pDC5 16-f sequencing (see Figure 3).

2.2.2 Construction of PDC 516-CD40-IgG: The CD40 fragment was connected with the linear vector of pDC5 16-IgG, and the correctness of the CD40 insertion sequence was confirmed by sequencing with primer PDC 516-F.

2.2.3 construction of PDC 5 16-CD40-IgG-GFP plasmid: GFP fragment was connected with pdc516-cd40-IgG linear vector, and the correctness of GFP insertion sequence was confirmed by sequencing pdc516-r. The fusion product of CD40-IgG-GFP containing restriction sites was 200 1bp, encoding 667aa (see Figure 4).

2.3 expression plasmid of recombinant plasmid pDC5 16-CD40-IgG-GFP in eukaryotic cells was extracted, and the OD value was 0. 1257 by ultraviolet spectrophotometer. On the 4th day after transfection of 293 cells with Lipofectamine 2000, GFP expression was observed in sporadic cells. With the passage of time, the number of cells expressing GFP gradually increased, and around the 7th day, a large number of cells showed green fluorescence under the fluorescence microscope (see Figure 5).

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