Creating a E.Coli Stock Culture

Before you begin

1. Make LB media as per the protocol titled Media Preparation

Once you have this

2. Transfer 25mL of LB media in to a sterile (autoclave) 250 mL erlenmeyer flask using a sterile Falcon Transfer Pipet

3. Inoculate the flask with enough stock culture(Glycerol stock obtained from a lab mate/purchased) to create a final OD600 of 0.025

4. Make note to have 1:10 ratio of liquid to air in your inoculated flask for aerobic growth of E.coli

5. Incubate the flask at 37C and a shaker speed of 100-150 rpm

6. Over every 30 min take an OD600 reading to track the growth of the E.coli cells in your flask

7. When the cells hit Stationary phase (OD600 greater than or equal to 10 in 1ml of culture) the culture is ready to make stock tubes for storage

8. At this point, take 1ml of culture and add 0.5ml of glycerol in a 2ml sterile tube, create 10-15 of these 2ml glycerol stock tubes