Product name	UltraReaction 3.0 Efficient Transfection Reagent
Catalog no	FXP135-010
Size	1 ml

Product Introduction

UltraReaction 3.0 is a new type of non liposome polymer transfection reagent that is used for efficient transfection in vitro with low cytotoxicity. It is suitable for culture media containing serum, especially for DNA transfection of large amounts of highly expressed proteins, high throughput screening of cDNA chips and RNA, as well as SiRNA. UltraReaction 3.0 can efficiently enrich DNA and ingest DNA into the cell through endocytosis, featuring strong binding and protection ability to DNA and low toxicity. Scope and characteristics of application: Compared with the most commonly used transfection reagents, the transfection efficiency is higher. 2. It can be used for both instantaneous and stable transfection. 3. Low cytotoxicity and the ability to increase protein expression through transient transfection. Both serum-containing and serum-free media can be used, unaffected by changes in the medium. Instructions for use: Although the following transfection methods have been fully validated and this reagent has high transfection efficiency, it is still recommended that researchers optimize the conditions for each type of cell, usually considering the following issues: 1. Cell density: Most adherent cells have a density between 50-70%, making them suitable for transfection. Determine the optimal cell density for transfection as the maximum efficiency for each cell type and maintain the reproducibility of density in all experiments. 2. Culture environment: Serum containing culture medium has no effect on UltraReaction 3.0 transfection. 3. This product is stored at -20 ℃. It is recommended to pack and freeze it before use to avoid the impact of repeated freezing on the transfection reagent. 4. DNA purity and concentration requirements: It is recommended to use high-purity and sterile DNA prepared by ion exchange columns. Removing endotoxin contamination in DNA is a key step in ensuring maximum transfection efficiency. Taking HEK293 cells as an example, the optimal DNA transfection concentration for 24 well plates is 0.5 μ g of DNA per well. The standard ratio of UltraReaction 3.0 to DNA is UltraReaction 3.0: DNA=3:1, which means that 3 μ l of UltraReaction 3.0 corresponds to the addition of 1 μ g of DNA. However, it is recommended that customers optimize the range according to experimental needs, that is, adjust the amount of UltraReaction 3.0 added (2-8 μ l) based on the amount of DNA per 1 μ g. (The standard ratio and dosage of UltraReaction 3.0 and RNA are the same as DNA) Please refer to Table 1 for specific amounts of DNA or RNA usage: Table 1: Recommended transfection conditions for UltraReaction 3.0 Operation method: Instantaneous transfection: Taking a 24 well plate as an example, please refer to Table 1 for the amount of DNA used: 1. Prepare cells to be transfected: Inoculate them into a 24 well plate at a density of 5x105/well for suspended cells and 5x104/well for adherent cells, and incubate overnight at 37 ℃; 30 minutes before transfection, replace the cells to be transfected with fresh culture medium. 2. Prepare UltraReaction 3.0/DNA complex: Dissolve 0.5 μ g DNA in 25 μ l serum-free medium, vortex shake and mix well; Add 1.5 μ l UltraReaction 3.0 to 25 μ l serum-free medium, vortex shake well, add the latter to the former, immediately vortex shake well for 10 seconds, and incubate at room temperature for 10 minutes. 3. Transfection: Add the UltraReaction 3.0/DNA complex mentioned above to each well of cells (containing 450 μ l of culture medium), and the volume of UltraReaction 3.0/DNA complex accounts for about 1/10 of the total volume. Gently shake the 24 well plate until the composite is evenly dispersed. Incubate at 37 ° C for 24-48 hours. If necessary, fresh culture medium can be changed after 6 hours of transfection. Stable transfection: Transfect according to the instantaneous method mentioned above. After 24 hours of transfection, the cells were passaged into selective culture medium at a ratio of 1:10 or higher. During the transfection process, an empty control group (containing only UltraReaction 3.0 and not DNA) should be set up. Regularly replace with new screening media to maintain cell growth and passage in selective media. After 1-2 weeks of transfection, most of the cells were killed, and the remaining cells that could survive and grow in selective culture medium were stably expressing the transfected plasmid, i.e. target cells that were stably integrated into the cell genome. Collection can only be carried out after the stable expression of transfected plasmids has reached a certain number of cell proliferation.

matters needing attention

1. Using high-purity DNA (A260/A280 ratio should not be less than 1.8), and sterile and endotoxin free is more conducive to achieving higher transfection efficiency. 2. Before transfection, ensure that the cells are in a good growth state and have the necessary cell density. 3. Inefficient complexes (UltraReaction 3.0/DNA complexes), the ratio of UltraReaction 3.0/DNA complexes, DNA purity and concentration, cell density, mycoplasma contamination, etc. may all cause low transfection rates. So before transfection, please optimize the conditions first. 4. Transfected genes carrying toxicity, poor incubation conditions, poor DNA quality, and low cell density can all cause high toxicity to cells. 5. After use, please immediately cover the lid to avoid prolonged exposure to the air, which may affect the transfection efficiency. For your safety and health, please perform transfection in a cell culture room that meets cleanliness requirements. During the operation, please wear laboratory clothes, disposable gloves, masks, and sterile hats.