İçindekiler
- 1 How do you prepare glycerol stock for bacteria?
- 2 What is the purpose of a glycerol stock?
- 3 What should be the concentration of glycerol stock?
- 4 How do you get 50 percent glycerol?
- 5 How do I start a culture from glycerol stock?
- 6 How do you make a 30% glycerol solution?
- 7 Does glycerol need to be autoclaved?
How do you prepare glycerol stock for bacteria?
Bacterial Glycerol Stocks
- Put 0.5ml bacterial culture in a sterile eppendorf tube.
- Add 0.5ml of sterile 80% (v/v) glycerol soution.
- Freeze on dry ice or directly into –70oc .
- Store at –70oC. Cells are best for about 4-6 months, but will probably work ok for a whole year.
What is the purpose of a glycerol stock?
However, if you want to store bacteria for a longer time, you will need to establish glycerol stocks. The addition of glycerol stabilizes the frozen bacteria, preventing damage to the cell membranes and keeping the cells alive. A glycerol stock of bacteria can be stored stably at -80°C for many years.
What should be the concentration of glycerol stock?
I use a 40% final concentration of glycerol. For this I mix equal amounts of bacterial culture and 80% glycerol (giving a final 40%). You should keep the cell-glycerol mixture at room temperature for half hour to allow the cells to incorporate glycerol.
How do you get 50 percent glycerol?
Make a 50% glycerol solution by diluting 100% glycerol in dH 20. Using snap top tubes is not recommended as they can open unexpectedly at -80°C. To recover bacteria from the glycerol stock, open the tube and use a sterile loop, toothpick or pipette tip to scrape some of the frozen bacteria off the top surface.
How do I start a culture from glycerol stock?
Start by adding a ~20 microliters of your Glycerol stock to 3 ml of a nutrient rich broth such as SOC and shake it at 37 celsius for ~8 hours. Then add the starter culture to 250 ml of LB with a selective antibiotic that your plasmid has resistance to.
How do you make a 30% glycerol solution?
Prepare a solution of 30% glycerol (v/v) by mixing 30 ml of glycerol with 70 ml of water. Transfer the solution to a screw cap glass bottle and sterilize by autoclaving at 121°C for 15 min.
Does glycerol need to be autoclaved?
Sterilize glycerol by autoclaving for 20 minutes at 15 pounds per square inch (psi) (1.05 kg/cm2) on liquid cycle. To 1.5 ml of bacterial culture, add 0.5 ml of the sterile glycerol in a labeled storage tube (final glycerol concentration of 15%).