Opt™G418

G418 is an aminoglycoside antibiotic commonly used as a resistance screening reagent in molecular genetic assays. The screening concentration depends on the cell type used, for example:

When screening mammalian cells, it is recommended to start with a screening concentration of 400 micrograms per milliliter. ‌

When screening plant cells, it is recommended to start with a screening concentration of 10 micrograms per milliliter.

When screening yeast cells, it is recommended to start with a screening concentration of 500 micrograms per milliliter.

The optimal screening concentration is usually determined through a kill curve, which involves gradually increasing the concentration of G418, observing cell death, and finding the minimum G418 concentration that can kill all cells. For example, when screening CHO cells, the recommended concentration range for G418 is 200-2000 micrograms per milliliter. ‌

In practical operation, it may be necessary to determine the specific optimal screening concentration through experiments, as the dosage of G418 may vary under different cell types and growth conditions. For example, when screening CHO cells, the recommended concentration range for G418 is 200-2000 micrograms per milliliter, while the recommended concentrations for screening plant and yeast cells are 10-100 micrograms per milliliter and 500-1000 micrograms per milliliter, respectively.

1. Preparation of screening medium: Determining the optimal screening concentration of G418 requires consideration of cell type, growth conditions, and other environmental factors, and determining the specific concentration range through experiments. Cells should be cultured with different concentrations of G418 before undergoing lentiviral infection

2. Resuscitation of cells: Transfer the cells for 2-3 passages, wait for stable cell growth, and then subculture and count. Approximately 2000 cells per well are seeded in a 24 well culture plate

3. Taking mammalian cell screening as an example:

1. Dilute G418 solution with culture medium to 0 ug/ml, 400 ug/ml, 500 ug/ml, 600 ug/ml, 700 ug/ml, 800 ug/ml, 900ug/ml, 1000ug/ml, 1100ug/ml, 1200ug/ml on a 24 well plate, with 1 ml of culture medium per well (complete culture medium containing G418), and 3-4 replicates per concentration

2. Change the medium after 12 hours, remove the culture medium from the culture wells, wash once with PBS, add different concentrations of screening medium to each well, change the screening medium every other day, and culture for 10-14 days, based on the lowest cell death concentration.

Start infecting cells after determining the concentration of G418:

3. Spread the cells that need to be infected in a 6-well plate (200000/well), transfect the cells the next day, and replace with fresh normal culture medium after 6 hours. Add the optimal screening concentration G418 medium 24 hours after transfection, and change the medium the next day.

4. After changing the medium once or twice (overnight cultivation after changing the medium), when the cells reach 50% -80%, aspirate all the culture medium, centrifuge at 3000-4000rpm, discard the supernatant, add twice the volume of fresh optimal screening concentration culture medium, filter at 0.22um, mix well at 4 ℃ and set aside.

When a large number of cells die after about 6 days of cultivation, the adaptive culture medium prepared in 5 can be used instead. Alternatively, serum concentration can be increased for cultivation, such as using 10% serum instead of 20% serum.

6. After 10 days of cultivation, reduce the concentration of G418 by half and maintain the screening pressure.

7. After screening for about 14 days, resistant xx cells can be observed, and the cells can be cultured and expanded using monoclonal method or by scraping off negative clones

Note: The product is only for scientific research use