Calcium phosphate - DNA coprecipitation method:

When the nucleic acid is in the form of a calcium phosphate-DNA coprecipitate, the DNA can be attached to the cell surface to facilitate the ingestion of the cells, or into the cell through the cleavage of the cell membrane lipid phase, and the DNA entering the cell is only 1 % to 5% can enter the nucleus, of which less than 1% of DNA can be integrated with cellular DNA, stably expressed in cells, and the frequency of gene transduction is about 10-4. This technique can be applied to any DNA. Introduction of mammalian animals for transient or long-term transformation. This method is the most commonly used and preferred method for adherent cell transfection.

1, dosing

(1) 2 x HBS 1.63 g NaCl

1.19g Hepes

0.023g Na2PO4.2H2O

Add water to 100ml pH7.1 and store at 4°C

(2) 2mmol/L CaCl2 filtration sterilization

(3) TE: 0.1mmol/L EDTA

1mmol/L Tris-HCL PH8.0

(4) G418 (neomycin G418) solution: 1g G418 dissolved in 1mmol / L Hepes solution, add H2O to 10mL filter sterilization 4 ° C preservation.

(5) G418 selection medium: G418 was prepared with DMEM containing 10% fetal bovine serum, and the concentration of G418 was 200-800 mg/L.

Note: Pre-test the recipient cells first, the concentration is the lowest concentration that can kill more than 50% of the cells within 10 to 14 days.

2. Operation steps [Method 1]:

(1) Donor DNA preparation:

The method was extracted by the DNA extraction method described above and dissolved in TE, 40 mg/L.

(2) Culture of recipient cells:

Studying oncogene transfer should select animal cell lines that do not contain human Alu sequences as recipient cells. For example, mouse NIH3T3 embryo fibroblast cell line, etc., the cells have a certain tendency to spontaneous transformation, generally inoculated cells one day before transfection, inoculation density 2 × 104 / cm2, with DMEM containing 10% fetal bovine serum, 37 ° C, 5% CO2 culture, until the cells accounted for 50 ~ 70% of the bottom area, for transfection experiments.

(3) Preparation of DNA-calcium phosphate precipitate

1 The donor cell DNA and the PSV2-neo plasmid vector DNA were prepared into a 40 mg/L DNA solution using TE, and a gene neo plasmid DNA solution (20 mg/L) 220 μl and 2×HBS were added to 200 μl of the donor cell DNA solution. 250μl (PSV2-neo is 1~2mg/L usage).

2 Take 500μl of the above DNA solution into the silicified tube, slowly add 3.1ml, 2mol / L CaCl2 and mix for 30 seconds.

3 Then immediately mix and spin, let stand for 30 minutes at room temperature, after the solution is slightly turbid, after blowing, it is used to transfect recipient cells.

(4) Transfected receptor cells

1 Recipient cells that have accounted for 50-70% of the bottom of the bottle in the logarithmic growth phase, and replace the fresh medium with 5 ml (25 ml culture flask) 4 hours before transfection.

2 Pipette 0.5mL of DNA-calcium phosphate precipitate and shake it in a cell bottle containing 5mL of culture solution.

3 culture at 37 ° C 5% CO2 for 24h or longer, so that the cells fully inhaled DNA-calcium phosphate crystal particles.

4 Replace the fresh medium and continue to culture for 24 hours to induce the expression of the transfected gene.

5 Replace the concentration of 800mg/L G418 selection medium for screening. At the same time, there are control cells that have not been transfected.

6 Culture for about 3 to 5 days, most of the control cells died. At this time, the transfected cells were replaced with G418 selection medium at a concentration of 200 mg/L, and the selection medium was changed every 3 to 4 days.

After 2 weeks, the control cells died. In the transfected cell bottle, drug-resistant cell clones appeared. After they were enlarged, they were cloned and expanded, and the transformed cell lines were established and further identified.

In this experiment, PSV2-neo, DNA and foreign DNA were co-transfected into recipient cells, so that the recipient cells can obtain the resistance of neomycin (neo) gene, so that even if the oncogene does not have obvious "transformation". The anti-neo marker of the transferred exogenous gene can also be detected. It is also possible to use the receptor cell introduced by the neo gene to select and transform the transformed cell to establish a cell strain by G418 selection. Objective, you can introduce PSV2-neo DNA into the recipient cells by simply extracting the genomic DNA extracted from the cancer cells.

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