ICSynBio:Streaking Culture

If you have a glycerol stock or stab culture of bacteria and need to purify plasmid DNA from it, you will want to isolate an individual clonal population (single colony) of bacteria from this stock. Using a single colony from a freshly streaked agar plate to inoculate a bacterial culture for DNA purification will minimize the chance of having a mixture of plasmids in your purified DNA. This protocol explains how to isolate a single bacterial colony by streaking it onto an LB agar plate.

Equipment

• Sterile toothpicks or wire loop
• Bunsen burner (or other small flame source)
• Incubator
• Marker

Reagents

• LB agar plate (with appropriate antibiotic)
• Bacterial stab

Methods

1. Obtain an LB agar plate with appropriate antibiotic.

2. Label the bottom of the plate with the plasmid name and the date. It is also a good idea to add the antibiotic resistance and your initials. Labeling within a laboratory setting is important for organization, and it is recommended that you keep a standard labeling system for all your objects/solutions.

3. Sterilize your lab bench by spraying it down with 70% ethanol and wiping it down with a paper towel. Maintain sterility by working near a flame or bunsen burner.

4. Obtain the approrpriate bacterial stab or glycerol stock.

5.Using a sterile loop, pipette tip or toothpick, touch the bacteria growing within the punctured area of the stab culture or the top of the glycerol stock.

6. Gently spread the bacteria over a section of the plate, as shown in the diagram above, to create streak #1.

7. Using a fresh, sterile toothpick, or freshly sterilized loop, drag through streak #1 and spread the bacteria over a second section of the plate, to create streak #2.

8. Using a third sterile pipette tip, toothpick, or sterilized loop, drag through streak #2 and spread the bacteria over the last section of the plate, to create streak #3.

9. Incubate plate with newly plated bacteria overnight (12-18 hours) at 37 °C.

10. In the morning, single colonies should be visible. A single colony should look like a white dot growing on the solid medium. This dot is composed of millions of genetically identical bacteria that arose from a single bacterium. If the bacterial growth is too dense and you do not see single colonies, re-streak onto a new agar plate to obtain single colonies.

11. Once you have single colonies, proceed to Recovering Plasmid DNA