How long does blasticidin take to kill cells?
Table of Contents
10-14 days
Replenish the selective media every 3-4 days, and observe the percentage of surviving cells. Determine the appropriate concentration of Blasticidin that kills the cells within 10-14 days after addition of the antibiotic.
How long does it take for hygromycin selection?
Ideally, control cells should die within one week after addition of the antibiotic, allowing colonies of resistant cells to form within 10–14 days. Hygromycin B is added to the culture medium at a concentration that varies with the cell type transfected.
What concentration of hygromycin is used for selection?
Gibco® Hygromycin B is used as a bacterial selection antibiotic in the concentration range of 200–500 µg/mL. This product is supplied as a 50 mg/mL solution.
What is a killing curve?
A kill curve is a dose-response experiment where the cells are subjected to increasing amounts of antibiotic to determine the minimum antibiotic concentration needed to kill all the cells over the course of one week.
How fast is blasticidin selection?
Typically, mammalian cells are sensitive to blasticidin concentrations of 1-10 µg/ml, and bacteria to 25-100 µg/ml. Cell death induced by blasticidin occurs rapidly, allowing for selection of transfected cell lines carrying a blasticidin resistance gene within one week.
How long does blasticidin last in media?
Blasticidin solution is stable for 9 months at −20°C to −5°C. Medium containing Blasticidin can be stored at 4°C for up to 2 weeks. On thawing, use what you need and discard the unused portion. For selection of Blasticidin-resistant E.
How does hygromycin selection work?
Hygromycin B has been used to select transfectants in a wide variety of cells including bacteria, protozoans, yeast, fungi, plants, and mammalian cells. Resistance to Hygromycin B is conferred by a gene coding for a phosphotransferase that phosphorylates Hygromycin B, thereby inactivating it.
Is hygromycin light sensitive?
The molecules of hygromycin are still light sensitive, no matter how much is there… you need to protect it from light.
What is hygromycin used for?
Hygromycin B is an aminoglycoside antibiotic not used to treat infections in humans and has antimicrobial activity against a wide range of microorganisms. Vectors with four replication origins are represented, with potential applications including general cloning, allelic replacement, and transcriptional analysis.
What is hygromycin resistance gene?
The resistance gene is a kinase that inactivates hygromycin B through phosphorylation. Since the discovery of hygromycin-resistance genes, hygromycin B has become a standard selection antibiotic in gene transfer experiments in many prokaryotic and eukaryotic cells.
Is neomycin same as G418?
G418 is an analog of neomycin sulfate, and has similar mechanism as neomycin. G418 is commonly used in laboratory research to select genetically engineered cells .
What is G418 selection?
Gibco® Geneticin® Selective Antibiotic (G418 Sulfate) is used as a selective antibiotic in the concentration range of 100 – 200 µg/ml for bacteria, or 200 – 500 µg/ml for most mammalian cells. This product is supplied as a 50 mg/ml solution in water. We offer a variety of cell culture antibiotics for your convenience.
How much hygromycin do you use for puromycin kill curve?
If using hygromycin, follow the same protocol as for the Puromycin Kill Curve but use 0, 50, 100, 200, 400, and 800 μg/ml hygromycin in six different wells. If you are using a construct with a neomycin selection marker, you need to test for resistance to geneticin (G418).
How do you do a puromycin selection experiment?
Mix and place the cells at 37°C in a CO 2 incubator. Grow cells under standard conditions for 72 hours. Count viable cells, and determine the lowest concentration of drug that kills at least 95% of cells in 3-5 days. Use this concentration at the puromycin selection step during your experiment.
How to test for neomycin resistance to geneticin (G418)?
If you are using a construct with a neomycin selection marker, you need to test for resistance to geneticin (G418). Use the same protocol as for the Puromycin Kill Curve, but use a range of concentrations between 400-800 μg/ml. Thanks for your feedback.
What is the kill curve procedure?
Kill curve procedure To express a recombinant protein of interest in mammalian cells, a mammalian expression vector containing the hyg or hph gene must be introduced into the mammalian host cell line. The hyg or hph marker gene allows for selection of stable transfectants using hygromycin B resistance.