Introduction of cell transfection experiment
First, the purpose of the experiment
Learn and master the main method of introducing foreign genes into eukaryotic cells - liposome-mediated transfection. To understand the general method of entry of foreign genes, observe the expression of foreign proteins (green fluorescent protein), and prepare experimental materials for staining.
Second, the experimental principle
The top panel is a schematic representation of liposome-mediated transfection, which shows the general process of entry of a foreign plasmid into a cell.
There are four main methods for entry of foreign genes into cells: electroporation, calcium phosphate and liposome-mediated methods, and virus-mediated methods. Electroporation is a short-term temporary perforation on a cell to allow foreign plasmids to enter; the calcium phosphate method and the liposome method use different carrier materials to carry a plasmid to allow foreign genes to enter cells by direct membrane or membrane fusion; The virus method uses a virus that encapsulates a foreign gene to infect cells to enter the cell. However, due to the strict control and difficulty of the experimental conditions of the electroporation method and the calcium phosphate method, the preliminary preparation of the virus method is complicated and may have a great influence on the cells; therefore, for many common cell lines, the general transient transfection method is now available. The liposome method is often used.
The most important thing to use lipofection is to prevent its toxicity. Therefore, the ratio of liposome to plasmid, cell density, length of transfection and serum content in the medium are all important issues affecting transfection efficiency. The appropriate transfection conditions for experimental exploration have a great effect on the improvement of efficiency.
The top panel is a schematic representation of the structure of the cationic component of the liposomes employed in this experiment.
The liposome used in this experiment is Promega's TransFast liposome reagent, which is a mixture of cationic liposome and neutral liposome. It is a transfection reagent optimized for 293T cells used in this experiment. .
Third, experimental materials and equipment
1, material
293T cell
MyoD expression plasmid and EGFP expression plasmid
DMEM medium streptomycin / penicillin (double antibody)
FCS (calf serum)
PBS (phosphate buffer solution)
Trypsin/EDTA Digestion Transfection Reagent (TransFast)
2, equipment
20ul/200ul/1ml micro pipette and Tip head alcohol lamp waste liquid cylinder blood cell counting plate vortex oscillator constant temperature water bath desktop centrifuge
35mm culture dish transfection tube
15ml centrifuge tube for observation with inverted microscope fluorescence microscope and CCD
Fourth, the experimental steps
1, cell passage
(1) Test preparation: 200 ul / 1ml Tip head each box (all items need to be autoclaved), alcohol cotton balls, waste liquid tank, test tube rack, micro pipette, marker pen, culture dish, centrifuge tube.
(2) Discard the medium in the Petri dish and wash twice with 1 ml of PBS solution.
(3) Add 1 ml of Trypsin solution to the Tip head and digest for 1 minute (37 ° C, 5% CO 2 ). The wall of the culture flask was patted by hand and the cells were observed to completely fall off the wall.
(4) Stop the reaction by adding 1 ml of serum-containing medium.
(5) Use the Tip head to breathe multiple times to completely disperse the cells.
(6) The culture solution was placed in a centrifuge tube and centrifuged at 1000 rpm for 5 min.
(7) Resuspend the cells with the culture medium. After the cells are counted, select 0.8× 10 6 cells and add a 35 mm culture dish.
(8) Add a suitable volume of complete culture solution to the centrifuge tube, mix the cells, and gently add to the culture dish to make it evenly distributed.
(9) Transfer the culture dish to a CO 2 incubator and transfect the next day.
2, cell transfection
(1) Preparation of transfection reagent
A. Add 400 ul of denuclease water to the tube and shake for 10 seconds to dissolve the lipid.
B. Store the reagent at -20 °C after shaking, and shake it before use.
(2) Select the appropriate mixing ratio (1:1-1:2/liposome volume: DNA mass) to transfect the cells. Add a suitable volume of serum-free medium to a transfection tube. Add DNA of the appropriate mass of MyoD or EGFP, shake it and then shake it again by adding the appropriate volume of transfection reagent.
(3) Leave the mixture at room temperature for 10-15 minutes.
(4) Aspirate the medium in the plate and wash once with PBS or serum-free medium.
(5) Add the mixture and return the cells to the incubator for one hour.
(6) After the time, decide whether to remove the mixed solution according to the cell type, and then continue to culture for 24 to 48 hours by adding the complete medium.
3. The second cell passage
(1) At 24 hours after transfection, the experimental results were observed and the expression of green fluorescent protein was recorded.
(2) Cell passage was again performed, and the cells were re-pulverized into a Petri dish according to a suitable density of 0.8× 10 5 cells/35 mm culture dish.
(3) After 24 hours of culture under normal conditions, it is fixed according to the dyeing requirements.
Transfection conditions can be optimized by reference to the TransFast instruction manual.
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