Product Introduction:

This product is hygromycin B dry powder, used for cell culture experiment after dissolution filtration to remove bacteria. Recommended solvent water, PBS solution.

Description:

Hygromycin B, an aminoglycoside antibiotic metabolized by Streptomyces hygroscopicus, inhibits protein synthesis by interfering with 70S ribosome translocation and inducing misreading of mRNA templates. This kills prokaryotic (e.g., bacteria), eukaryotic (e.g., yeast, fungi) and higher mammalian eukaryotic cells.

Escherichia coli (hyg or hph) -derived hydthromycin resistance gene encodes hydthromycin B phosphotransferase, which converts hydthromycin B into a non-biologically active phosphorylated product, thus playing a detoxification role. In response to this principle, hygromycin B is a very useful selective marker for screening and maintenance of cultured prokaryotic or eukaryotic cells successfully transfected with hygromycin resistance genes. In addition, due to differences in mode of action, it is often used in combination with G418 (Cat: G8161), Zeocin (Cat: Z8020), and Blasticidin S (Cat: B9300) for the selection of double-resistant positive cell lines. Hydomycin B can also be used as an antiviral agent because it selectively penetrates into cells that enhance permeability due to viral infection and has the effect of inhibiting translation. It can also be mixed into animal feed to repel insects.

Use and synthesis method:

Inhibitors of protein synthesis, which can be used in biochemical studies such as plant cell culture.

Usage method

1. Commonly used screening concentration

Note: The working concentration of hygromycin B for screening stable strains varies depending on cell type, medium, growth conditions, and cell metabolic rate, with a recommended concentration of 50-1000μg/mL. For the first use of the experimental system, it is recommended to establish a kill curve, that is, a dose-reactivity curve, to determine the optimal screening concentration.

In general, mammalian cells 50-500μg/mL; Bacterial/plant cells 20-200μg/mL; Fungi 300-1000μg/mL.

2. Establishment of killing curve

Note: In order to screen cell lines for stable expression of the target protein, it is necessary to determine the minimum concentration of the antibiotic that can kill the untransfected host cells, which can be achieved by establishing a kill curve (dose-response curve), with at least 5 concentrations selected.

1) Day 1: Untransformed cells were spread on a suitable culture plate with a cell density of 20-25% and cultured overnight at 37℃ in CO2; Note: For cells that require higher density to detect viability, the amount of inoculation can be increased.

2) Set the concentration gradient within the appropriate range according to the cell type. In the case of mammalian cells, 50,100,250,500,750,1000 μg/mL can be set. Dilute the mother solution to 5 mg/ml with deionized water or PBS buffer at the ratio of 1:10, and then dilute to the corresponding concentration of the working solution according to the following table.

3) The second day: Replace the old medium with a freshly prepared medium containing the corresponding concentration of drugs. Three parallel holes for each concentration.

4) Replace the new drug-containing medium every 3-4 days.

5) Live cell counts are performed on a fixed cycle (e.g., every 2 days) to determine the appropriate concentration to prevent the growth of untransfected cells. Select the lowest concentration that will kill the vast majority of cells within the desired number of days (usually 7-10 days) as the working concentration for stable transfection cell screening.

3. Screening of stable transfected cells

1) 48h after transfection, the cells were passed with a screening medium containing appropriate concentration of hygromycin B (direct passage or diluted passage).

Note: Antibiotics are most effective when cells are actively dividing. When the cells are too dense, they become less efficient. In order to obtain a better screening effect, it is best to dilute the cells to the abundance of no more than 25%.

2) Change the drug-containing screening medium every 3-4 days.

3) The formation of cell clones (colonies) was observed and evaluated after 7 days of screening. Colony formation may take another week or more, depending on the host cell type, transfection, and screening effectiveness.

4) 5-10 resistant clones were selected and transferred to 35mm cell culture plates and continued to be cultured with drug-containing screening medium for 7 days.

5) Replace the normal culture medium after cultivation.

Note:

1) hygromycin B resistance gene (hyg or hph) except from E. Coli. In addition, other strains have been found, including Streptomyces hygroscopicus and Klebsiella pneumoniae.

2) This product is a toxic compound, avoid contact with skin and eyes, please use caution.

3) For your safety and health, please wear a lab coat and disposable gloves.

Note：Product information may be optimized and upgraded. Please refer to the actual label information for accuracy.