Summary: G418 (Geneticin) Selection. G418 is an aminoglycoside antibiotic produced by Micromonospora rhodorangea. G418 blocks polypeptide synthesis by inhibiting the elongation step in both prokaryotic and eukaryotic cells. Resistance to G418 is conferred by the neo gene from Tn5 encoding an aminoglycoside 3'-phosphotransferase, APT 3' II. G418 is commonly used to select cells which have been transfected with a plasmid expressing the neo gene. In general, G418 concentrations of 400 mg/L are used for selection and 200 mg/L for maintenance for mammalian cells. However, optimal concentration for resistant clone selection in mammalian cells should be tested for a specific cell line. To do this, antibiotic titration is done using antibiotic concentrations ranging from 100 mg/L up to 1400 mg/L to determine the killing curve. Resistant clones selection may take 1 to 3 weeks.
- Kill/Death Curve Guide (PROVOST & WALLERT RESEARCH MN STATE UNIVERSITY MOORHEAD)
Protocol for preparation of G418 and Puromycin, and for determining kill curve.
Added: Sat Feb 16 2013, Hits: 771, Reviews: 0
Determining Kill Curve
(Bioinformatics Courses at the Bioinformatics Centre, National University of Singapore)
Before doing a transfection experiment, it is important to determine the concentration of selection reagent required for efficient selection. This is usually achieved by doing a kill curve, which is basically growing cells in various concentrations of the selection reagent.
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G418 Sulfate Preparation
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Preparation of G418
(S Cell Targeting Core Laboratory, John Hopkins University)
To produce a solution of G418 and hepes in the concentration of 500 mg of active G418 to 1 ml of hepes.
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Preparation of Neo (G418) Stock
Added: Sat Feb 16 2013, Hits: 274, Reviews: 0