Transfection of HEK293T cells by calcium phosphate method
Transfection of HEK293T cells by calcium phosphate method can be applied to (1) study the mechanism of transfection of mammalian cells and (2) genetic engineering.
Operation method
Calcium phosphate method
Principle
Conventional transfection techniques can be divided into two main categories, transient transfection and stable transfection (permanent transfection). In the former, the exogenous DNA/RNA is not integrated into the host chromosome, so multiple copy numbers can exist in one host cell, generating high levels of expression, but usually lasting only a few days, and is mostly used for the analysis of promoters and other regulatory elements. In general, superhelical plasmid DNA transfection is more efficient, and results are analyzed within 24-72 hours post-transfection (depending on a variety of different constructs), often using a number of reporter systems.
Materials and Instruments
293T cells Move 1. Cell spreading: Select 293T cells in good condition for passaging, 2-3x105 cells/35mm dish. 2. 20-24h later, let the cells grow to about 50-70% of the bottom of the bottle. 2. 20-24h later, when the cells have grown to about 50-70% of the bottom of the bottle, transfection will be carried out. Take 35mm dish as an example, use the following transfection system: ddw: 105ul plasmid: 2ug (e.g. 0.5ug/ul, i.e. 4ul) 2M CaCl2: 16.5ul 2M CaCl2: 16.5ul 2XHBS: 125ul Add the above four reagents to the eppendorf tube in the above order. Mix the first three first and add 2XHBS last. One method is to add 2XHBS drop by drop, blow until it becomes milky, then drop it evenly into the petri dish, shake gently and put it in the incubator, then change the liquid after 6-8h. Another method is to add 2XHBS and blow about 40 times (but this depends on the force and frequency of blowing, it is recommended to do a gradient experiment, blowing different times to see which time the transfection efficiency is high and then blow according to this number of times), then the steps are the same as the above, and then drop into the petri dish, gently shaking it well and then put it into an incubator, after 6-8h, change the liquid. Caveat 1. When the pH value of HBSP buffer was 6.70 and 7.10, the transfection of HEK 293T cells was not effective, the cell viability was low, and there were large granular objects; when the pH value was 6.96, the transfection was effective, the cell viability was high, and the blue-stained cells were obvious. 2. Unpurified lacZ-pShuttle plasmid transfection of HEK 293T cells showed low transfection efficiency and low cell viability, while the purified plasmid showed high transfection efficiency and obvious bluish stained cells. Common Problems 1. Glycerol shock at 6h after transfection was the most effective, and the transfection efficiency was 584.4% higher than that at 0h. 2. The best transfection effect was achieved when the glycerol shock time was 4.5 min, which increased the transfection efficiency by 130.8 % compared with that without glycerol shock. For more product details, please visit Aladdin Scientific website.
Sterile water ddw NaCl KCl Na2HPO4 glucose HEPES CaCl2
Eppendorf tubes Petri dishes Incubator