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Frequently Asked Questions

QUESTION: G-418 works on what cell types?
ANSWER: G-418 inhibits eukaryotic cell's protein synthesis. G-418 is toxic to bacteria, yeast, protozoans, higher plant, and mammalian cells.

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 ranges from 400 ug / ml for selection and 200 ug / ml for maintenance are required for mammalian cells. Plant cells require 25-50 ug / ml for selection and 10 ug / ml for maintenance and bacteria 8-16 ug / ml for selection.

QUESTION: Is G-418 the same as Geneticin®?
ANSWER: G-418 and Geneticin® are the same chemical structure. G-418 is the generic chemical while Geneticin® is a registered trademark brand of Invitrogen Corporation

QUESTION: What is the potency specification for G418 liquid and powder??
ANSWER: The potency specification is > 700 ug/mg. Small lot-to-lot variances do occur.

QUESTION: What gene confers resistance to G-418?
ANSWER: The neo / kan gene. This gene allows production of aminoglycoside 3'-phosphotransferase, which inactivates G418, Neomycin and Kanamycin by phosphorylation.

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: Can mammalian cells in culture divide in the presence of G418?
ANSWER: Yes. Cells can continue to divide up to two times in the presence of lethal doses of G418. The effect of G418 usually becomes apparent by two days.

QUESTION: How do I work with the reported potency of G418?
ANSWER: The potency reported is microbiological potency. It is a function of G418 inhibitory effect on B. subtilis ATCC6633. Potency is reported in mg/mg. To convert the potency reported on the powder to liquid potency divide the potency by 1000 and multiply this by the desired potency.

For example: If the potency reported on the powder is 750 ug/mg and you wish to make a solution of 400 mg/mL you would: 1000/750 x 400 mg/mL = 533.33 mg/mL

We recommend that a dose response curve be set for both sensitive and resistant mammalian cells. Refer to the Certificate of Analysis under ED50 Sensitive and ED50 Resistant for suitable concentrations of G418 to use for the curve. These concentrations are reported as dry weight. G418 is most effective against dividing cells. By permitting cell growth prior to the addition of G418, one will maximize the selection characteristics of the antibiotic for both sensitive and resistant cells.

Procedure:

To generate a stable cell line expressing your protein of interest, you need to determine the minimum concentration of antibiotic required to kill your untransfected host cell line. Test a range of concentrations (at least 6) to ensure that you determine the minimum concentration necessary for your cell line.
• 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.
• The next day, substitute culture medium with medium containing varying concentrations of the antibiotic (i.e., test 0, 50, 100, 200, 400, 800, 1000 ug/mL).
• Replenish the selective medium every 3-4 days.
• Count the number of viable cells at regular intervals to determine that appropriate concentration of antibiotic that prevents the growth of untransfected cells. Select the concentration that kills the majority of the cells in the desired number of days, generally 7-10 days

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 400 ug / ml for selection and 200 ug / ml for maintenance are required for mammalian cells. Plant cells require 25-50 ug / ml for selection and 10 ug / ml for maintenance and bacteria 8-16 ug / ml for selection.Your optimum concentration should be tested experimentally.

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