Protocol: G418 (Geneticin) Stock Solution:

 

Geneticin is an aminoglycoside related to Gentamicin and is toxic to bacteria, yeast, higher plants, mammalian cells, protozoans and helminths. The resistance genes are located on transposons Tn601(903) and Tn5 and are bacterial in derivation, but they can be expressed in eucaryotic cells. By introducing these genes into cells, resistance to Geneticin is conferred. It is used for the selection of transfected mammalian cells, yeast, dictyostelium, plant and bacteria.

Certain transfection techniques in molecular biology and cell culture require the use of antibiotics to select for transfected cells. Geneticin is an effective selecting agent utilized in killing procaryotic and eucaryotic cells. The mechanism of action occurs by inhibiting protein synthesis by binding to ribosomes of prokaryotic and eukaryotic cells, therefore killing the non-resistant cells. Due to this binding mechanism, cells will generally take several days to die. Resistance is conferred by the bacterial gene for aminoglycoside-3-phosphototransferase that can be expressed in eucaryotic cells. Cells may have variable resistance and can take up to one week to die; adherent cells may be more sensitive.

1. Weight 2gr of G418 (CAS: 108321-42-2 mw=692.71).

2. Add 10ml of sterile H2O. Dissolve completely.

3. Prewet a 0.22um syringe filter by drawing through 5-10ml pf sterile H2O and discard water.

4. Sterilize G418 Stock through through the prepared 0.22um syringe filter.

5. Stock may be kept at -20C for 1 year.