G4020： Hygromycin B (100mg/ml), 1ml
G4020： Hygromycin B (100mg/ml), 1ml
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Product Description/Introduction
Hygromycin B is an aminoglycoside antibiotic produced by the metabolism of Streptomyces suis. Hygromycin B kills prokaryotic (bacterial), eukaryotic (e.g., yeast, fungi), and higher mammalian eukaryotic cells by interfering with 70S ribosomal translocations and inducing misreading of mRNA templates and thereby inhibiting protein synthesis.
The E. coli-derived hygromycin resistance genes (hyg or hph), which encode hygromycin B phosphotransferases, detoxify hygromycin B by converting it into a biologically inactive phosphorylated product. In response to this principle, hygromycin B is a very useful resistance selection marker that can be used to screen and maintain in culture prokaryotic or eukaryotic cells that have been successfully transfected with hygromycin resistance genes. In addition, it is often used in combination with G418 for the selection of dual resistance-positive cell lines due to differences in the mode of action.
This product is a sterile solution of hygromycin B (50 mg/mL), which can be directly diluted with culture medium for use. The commonly used working concentration is 200-500 μg/mL.For first time use in experimental systems, it is recommended to determine the optimal screening concentration by establishing a dose-response curve.
Storage and Shipping Conditions
Ship with wet ice. Store at 2-8℃ for 24 months.
Assay Protocol / Procedures
1. Determination of a dose-response curve for hygromycin B:
For cells used for the first time, it is generally necessary to experimentally determine a dose-response curve (dose-response curve or kill curve) that is appropriate for your experimental system.
1) Day 1: 24-well plates were inoculated with untransfected cells at a density of 5~8×104cells/well, inoculating a sufficient number of wells for subsequent dose gradient experiments. Cells were cultured overnight in a cell culture incubator.
2) Day 2: Replace the cells after overnight incubation with freshly prepared screening medium containing different concentrations of hygromycin B (e.g. 0, 50, 100, 250, 500, 750, 1000 μg/mL, etc.), and continue the incubation in a cell culture incubator after replacing the medium. Next, the medium was replaced with fresh antibiotic-containing medium every 3-4 days.
3) Live cell counts were performed at fixed intervals to determine the appropriate concentration to block the growth of untransfected cells, and the lowest concentration that killed the majority of cells at 7-10 days was selected as the working concentration for screening of stably transfected cells.
2. Screening of stably transfected cells:
Stable expression strains can be screened after transfection of a plasmid containing the resistance gene or infection with a virus containing the gene.
1) Cells were transfected or infected for 48 h, and then cultured in fresh medium containing the appropriate concentration of hygromycin B.
Note: The antibiotic effect is most pronounced when the cells are in the active phase of division. If the cells are too dense, the effectiveness of the antibiotic will be significantly reduced, so it is best to keep the density of the cells to no more than 25%. It is recommended to make a control group of normal cells at the same time. After 48 h of transfection or infection, if the cells are too dense you can also re-inoculate the cells after digestion and incubate them overnight forhygromycin B screening.
2) Every 3-4 days, replace the medium with fresh medium containing antibiotics.
3) After 7 days of screening, 100% of the normal cells in the control group should be dead, and the surviving cells in the transfected group are those expressing the hygromycin B resistance gene. Then screen for polyclonal or monoclonal cells according to the experimental purpose.
4) The selected stable resistant clones continued to be maintained in screening medium containing the drug for 7-10 days, after which the normal medium was simply replaced.
Product Description/Introduction
Hygromycin B is an aminoglycoside antibiotic produced by the metabolism of Streptomyces suis. Hygromycin B kills prokaryotic (bacterial), eukaryotic (e.g., yeast, fungi), and higher mammalian eukaryotic cells by interfering with 70S ribosomal translocations and inducing misreading of mRNA templates and thereby inhibiting protein synthesis.
The E. coli-derived hygromycin resistance genes (hyg or hph), which encode hygromycin B phosphotransferases, detoxify hygromycin B by converting it into a biologically inactive phosphorylated product. In response to this principle, hygromycin B is a very useful resistance selection marker that can be used to screen and maintain in culture prokaryotic or eukaryotic cells that have been successfully transfected with hygromycin resistance genes. In addition, it is often used in combination with G418 for the selection of dual resistance-positive cell lines due to differences in the mode of action.
This product is a sterile solution of hygromycin B (50 mg/mL), which can be directly diluted with culture medium for use. The commonly used working concentration is 200-500 μg/mL.For first time use in experimental systems, it is recommended to determine the optimal screening concentration by establishing a dose-response curve.
Storage and Shipping Conditions
Ship with wet ice. Store at 2-8℃ for 24 months.
Assay Protocol / Procedures
1. Determination of a dose-response curve for hygromycin B:
For cells used for the first time, it is generally necessary to experimentally determine a dose-response curve (dose-response curve or kill curve) that is appropriate for your experimental system.
1) Day 1: 24-well plates were inoculated with untransfected cells at a density of 5~8×104cells/well, inoculating a sufficient number of wells for subsequent dose gradient experiments. Cells were cultured overnight in a cell culture incubator.
2) Day 2: Replace the cells after overnight incubation with freshly prepared screening medium containing different concentrations of hygromycin B (e.g. 0, 50, 100, 250, 500, 750, 1000 μg/mL, etc.), and continue the incubation in a cell culture incubator after replacing the medium. Next, the medium was replaced with fresh antibiotic-containing medium every 3-4 days.
3) Live cell counts were performed at fixed intervals to determine the appropriate concentration to block the growth of untransfected cells, and the lowest concentration that killed the majority of cells at 7-10 days was selected as the working concentration for screening of stably transfected cells.
2. Screening of stably transfected cells:
Stable expression strains can be screened after transfection of a plasmid containing the resistance gene or infection with a virus containing the gene.
1) Cells were transfected or infected for 48 h, and then cultured in fresh medium containing the appropriate concentration of hygromycin B.
Note: The antibiotic effect is most pronounced when the cells are in the active phase of division. If the cells are too dense, the effectiveness of the antibiotic will be significantly reduced, so it is best to keep the density of the cells to no more than 25%. It is recommended to make a control group of normal cells at the same time. After 48 h of transfection or infection, if the cells are too dense you can also re-inoculate the cells after digestion and incubate them overnight forhygromycin B screening.
2) Every 3-4 days, replace the medium with fresh medium containing antibiotics.
3) After 7 days of screening, 100% of the normal cells in the control group should be dead, and the surviving cells in the transfected group are those expressing the hygromycin B resistance gene. Then screen for polyclonal or monoclonal cells according to the experimental purpose.
4) The selected stable resistant clones continued to be maintained in screening medium containing the drug for 7-10 days, after which the normal medium was simply replaced.
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When can I expect my order to ship? Most orders are filled and shipped within 2-3 business days from the time they are received. Our standard shipping usually take 2-5 days. In case of a back order, we will immediately notify the customer by email. We also provide express shippping for time-sensitive deliveries. Email info@genelabx.com if you have any requirements.This is an answer
When can I expect my order to ship? Most orders are filled and shipped within 2-3 business days from the time they are received. Our standard shipping usually take 2-5 days. In case of a back order, we will immediately notify the customer by email. We also provide express shippping for time-sensitive deliveries. Email info@genelabx.com if you have any requirements.