HYGROMYCIN B FAQ'S Top 10
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6450 Lusk Blvd. Suite E102
San Diego, CA 92121
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Many researchers have foregone searching for new antibiotics and instead turned to older antibiotics who for various reasons never reached clinical application. A.G.Scientific emerged as a leading manufacturer and importer of Hygromycin B, an antibiotic used for gene selection. Over the past 15 years we have learned a lot about this Aminoglycosidic antibiotic. It was our success with a combination of fermentation & synthesis that has allowed us to build a customer base of researchers and catalog biochemical distributors worldwide. Questions on solution storage & what are the proceedures to determine toxic concentration are common to all customers of Hygromycin. So here we listed the top questions we have received from our customers. A Material and Safety (MSDS) document is also available for download at the bottom of the blog.
Hygromycin B FAQS (Frequently Asked Questions)
QUESTION: What is the stability of Hygromycin B solution?
ANSWER: Hygromycin B solution is stable for at least two years at +4°C and about one month at +37°C.
QUESTION: What color is Hygromycin B solution?
ANSWER: The color can vary from light yellow to a dark brown or caramel color. The concentrated Hygromycin solution tends to have a darker appearance.
QUESTION: What is the working concentration for selection?
ANSWER: The working concentration for the purpose of selection varies with cell type, media, growth conditions and cell metabolic rate. Recommended concentration for the selection of resistant cells is 25-1000 ug/ml. Commonly used concentrations for selection are 200 ug/ml for mammalian cells, 20-200 ug/ml for plant cells & bacteria cells and 200-1000 ug/ml for fungi. Your optimum concentration should be tested experimentally.
QUESTION: How can non-transfected cells escape antibiotic selection?
ANSWER: Cells can escape selection if the antibiotic is used at too low concentration or if the cell density on the plate is too high. Additionally, cells rapidly proliferating are killed faster than these, only slowly proliferating. Control cells should die within 5-7 days after addition of the antibiotic allowing colonies of resistant cells to form by 10–14 days.
QUESTION: How do I determine the Toxic Concentration?
ANSWER: Hygromycin B is added to the culture medium at a concentration that varies with the cell type transfected. A titration experiment for each cell type may therefore be performed to determine the amount of Hygromycin B needed to kill non-transfected cells. The working concentration for mammalian cell selection is normally between 50 ug/ml and 1mg/ml, Plant cells: 20-200 ug/ml, Bacteria: 20-200 ug/ml and Fungi: 200 ug-1mg/ml. Your appropriate concentration should be tested experimentally.
QUESTION: How do I perform a Dose Response curve?
ANSWER: To determine the minimum concentration of antibiotic required to kill your non-transfected host cell line.
A.) Test arranges of concentrations (5-6) to ensure that you determine the minimum concentration necessary for your cell line.
B.) Seed cells at approximately 20-25% confluency on the appropriate number of plates for each time plate and allow cells to adhere overnight. For cells that require higher densities for viability, increase the number of cells seeded.
C.) The next day, substitute culture medium with medium containing varying concentration of the antibiotic.
D.) Replenish the selective medium every 3-4 days.
E.) Count the number of viable cells at regular intervals to determine the appropriate concentration of antibiotic that prevents the growth of non-transfected cells. Select the concentration that kills the majority of the cells in the desired number of days, usually 7-10 days.
Interested in the complete HYGROMYCIN FAQ'S?
INCLUDED IS THE FOLLOWING:
- Consolidated FAQS gathered in a single document.
- Trouble Shooting Guide included.
- MSDS included in FAQs document.
- Selection & Maintenance tips.
- Information on performing Dose Response curve.
CLICK BELOW for complete HYGROMYCIN B FAQ'S
Sincerely, Chip Lindgren, Founder & President, A.G. Scientific, Inc.
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A.G. Scientific, Inc., an ISO 9001:2008 Company
6450 Lusk Blvd. Ste. E102,
San Diego, CA 92121, Phone +858 452 9925,
Fax +858 452 9926, www.agscientific.com
Personal email: chip@agscientific.com
Corporate email: info@agscientific.com
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