- 1 How much puromycin should I take for selection?
- 2 Why are antibiotics added to cell culture media?
- 3 How do you make a kill curve for antibiotics?
- 4 How do you treat cells with puromycin?
- 5 How does puromycin selection work?
- 6 How long does puromycin selection take?
- 7 Why is penicillin used in cell culture?
- 8 How is bacterial contamination treated in cell culture?
- 9 Why must antibiotics not be used very often in cell culture?
- 10 How long does Blasticidin take to kill cells?
- 11 How long does G418 take to kill cells?
- 12 What is G418 selection?
- 13 Does puromycin kill cells?
- 14 How long does media puromycin last?
- 15 How do you make a cell line stable?
How much puromycin should I take for selection?
Puromycin antibiotic ensures effective positive selection of cells expressing the puromycin -N-acetyl- transferase (pac) gene. In mammalian cells, the recommended working concentration range for puromycin is 0.5 – 10 µg/ml.
Why are antibiotics added to cell culture media?
Use of antibiotics in cell culture minimizes the loss of valuable cells, reagents, time and efforts due to contamination. Apart from preventing contamination, certain antibiotics also function as selection agents, used to select and establish transfected/genetically modified cells for research purposes.
How do you make a kill curve for antibiotics?
- Harvest healthy adherent cells either by using trypsin or by gentle cell scraping.
- Dilute the cell suspension in complete medium and seed each well of 96-well plate so as that the final volume is 100 ul.
- Typical cell density before antibiotic treatment in a 24-well plate:
How do you treat cells with puromycin?
A method we used most recently was to transfect cells as per normal procedure, then 24 hours later, treat with puromycin. You’ll need to do a Puromycin kill curve on your cells first to check what concentration of puromycin kills 100% of untransfected cells. Normally between 1-10ug/ml works.
How does puromycin selection work?
Puromycin is an aminonucleoside antibiotic produced by the bacterium Streptomyces alboniger. It inhibits protein synthesis by disrupting peptide transfer on ribosomes causing premature chain termination during translation. It is a potent translational inhibitor in both prokaryotic and eukaryotic cells.
How long does puromycin selection take?
Puromycin selection requires a minimum of 48 hours. Optimum effectiveness should be reached within 3-10 days. Assay transfected cells.
Why is penicillin used in cell culture?
The antibiotics penicillin and streptomycin are used to prevent bacterial contamination of cell cultures due to their effective combined action against gram-positive and gram-negative bacteria.
How is bacterial contamination treated in cell culture?
The common method for eliminating bacterial contamination is to supplement antibiotics into the medium. However, the antibiotics generally have their unique antibacterial spectra and no single antibiotic is effective against all bacteria.
Why must antibiotics not be used very often in cell culture?
Antibiotics are not the good Samaritans in animal cell culture. These chemicals affect the metabolism of the cultured cells and affect proliferation, differentiation and gene expression of the cells, thus hampering experiment results and questioning the validity of the same.
How long does Blasticidin take to kill cells?
Product Qualification. Blasticidin S HCl is lot qualified by performing a kill curve on Blasticidinsensitive and Blasticidin -resistant mammalian cell lines. Blasticidinsensitive cells should be killed at all concentrations tested (2.5-10 μg/ml) within 10 days after addition of Blasticidin.
How long does G418 take to kill cells?
3. Replace the G418 -containing medium every 2 – 3 days and examine cells for visual toxicity. Most non-transfected (non-resistant) cells will die within 10 days, leaving the transfected cells to expand.
What is G418 selection?
G418 (Geneticin): selection antibiotic, endotoxin tested, sterile reagent. G418 blocks polypeptide synthesis by inhibiting the elongation step in both prokaryotic and eukaryotic cells.
Does puromycin kill cells?
Puromycin is used in cell biology as a selective agent in cell culture systems. It is toxic to prokaryotic and eukaryotic cells. Puromycin acts quickly and can kill up to 99% of nonresistant cells within 2 days.
How long does media puromycin last?
Suggestions: 1) If you need to add the puromycin to your media, leave the media at room temperature to warm up from 4C storage instead of using the waterbath. This will keep it stable for ~ 3 weeks.
How do you make a cell line stable?
The protocol for generating stable cell lines requires several steps as shown below:
- Generate a kill curve to determine the optimal selection antibiotic concentration.
- Transfect cells with desired plasmid construct(s)
- Select and expand stable polyclonal colonies.
- Identify single clones by limited dilution and expansion.