Product Introduction:

Antibiotic selection: Cell culture tests, sterile reagents

Mechanism of action:

Blasticidin S is a nucleoside antibiotic from Streptomyces griseochromogenes that specifically inhibits protein synthesis in prokaryotes and eukaryotes by interfering with the formation of peptide bonds in ribosomes. Blasticidin S is used to screen transfected cells carrying bsr or BSD tolerance genes. Blasticidin has a rapid and potent mode of action, and very low concentrations of antibiotics can lead to rapid cell death.

Resistance genes:

Currently, three blasticidin tolerance genes have been cloned and sequenced, one of which was isolated from the producing bacterium Streptoverticillum sp. The other two are deaminase genes, including bsr isolated from Bacillus cereus and BSD isolated from Aspergillus terreus. The Bsr and BSD genes are the most commonly used screening markers for gene transfer experiments in mammalian and plant cells, as well as for E. coli.

Transportation and storage methods

wet ice packs are transported. After receiving the product, store it at -20 oC for at least 1 year to avoid repeated freezing and thawing.

Use and synthesis method:

Application concentration:

1) Escherichia coli

E. coli is slightly less sensitive to Blasticidin S, but inverters are tolerant to Blasticidin S and can be screened using low-salt LB medium (pH 8) with concentrations ranging from 50-100 μg/ml of Blasticidin S. High pH can increase the activity of Blasticidin S.

2) Mammalian cells

The working concentration of Blasticidin S in mammalian cells ranges from 1-50 μg/ml. The initial experiment suggested that the kill curve should be used to determine the optimal concentration.

- 25-100 μg/ml in bacteria

- 1-30 μg/ml in mammalian cells

Procedure (Screening of mammalian stable strain) :

Blasticidin S is usually used in a concentration of 10 μg/ml. Plasmids carrying bsr or BSD genes were transfected into cells and incubated in a normal growth medium containing Blasticidin S for screening stable transfected cell lines.

(1) 48h after transfection, the cells were transfected with a fresh medium containing a suitable concentration of Blasticidin S (note: Antibiotics work best when the cells are in the active division phase. The cell density is too high, and the antibiotic efficiency is reduced. The coverage rate of cells should not exceed 25%);

(2) Remove the medium every 3-4 days and add fresh medium containing antibiotics;

(3) Cell colony formation was detected 7 days later. Depending on the host cell type and transfection/screening efficiency, colony formation may take a week or more to increase.

(4) 5-10 tolerant clones were transferred to 35mm cell trays and added to selected media for 7 days. The cytotoxicity test was then performed.

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