In this exercise, you will learn how to obtain an isogenic E. coli cell culture from a frozen stock. This is important because storing plasmid DNA inside frozen E. coli cells is a popular method to preserve plasmid DNA. This method is like creating a backup copy of an important document. Once your plasmid is 'saved' in an -80°C freezer, you will be able to return to it at any time in the future.
Learning Objectives
Work safely and efficiently around an open flame.
Prepare, label and handle agar petri plates.
Identify, label and pick single bacterial colonies growing on a plate, and preserve a master plate.
Prepare, label and handle liquid cell cultures.
2.1: Streaking Bacteria
Materials
70% Ethanol Solution
3 Agar Plates
Streaking sticks
Bunsen Burner
Pre-streaked plate/Frozen Cell Sample
Note: If using Frozen Cell samples you will also need an ice box
Instructions
1. Wipe down your work area
2. Label the base (smaller side) of your plates (your name, bacterial strain, plasmid, date, lab bench). Place them upside down on your bench.
3. Ask your instructors for the pre-streaked plates that you will be using. If you are using frozen cell samples, fill your ice bucket with ice and retrieve a cell vial from your instructors
4. Turn on your flame
If working from a pre-streaked plate
5. Place the plate so that the lid is on the desk.
6. Retrieve a wooden stick (in your dominant hand) and run the end of it quickly through the flame to sterilize it
7. Lift up the bottom of pre-streaked plate (with your non-dominant hand) and use the stick to poke one of the isolate colonies on the plate. The bottom of your tip should show some slight color. Do not jab the agar
8. Close the pre-streaked plate
If working from a frozen cell sample
5. Unscrew the sample and place in the ice bucket
6. Retrieve a wooden stick (in your dominant hand) and run the end of it quickly through the flame to sterilize it
7. Dab the stick in the sample vial
8. Lightly place the lid back on the sample vial (you can tightly screw it back on when you are done with your work)
9. With your free hand, lift up the bottom of your freshly labelled plate. Gently streak cells across the agar surface (keep your streaking to one third of the plate)
10. Run your wooden stick through the flame to sterilize it before placing it in the biological waste bin
11. Retrieve a new wooden stick and drag it through the area you just streaked before spreading it out in another third of the plate.
12. Repeat steps 5-11 for your other plates
13. Close all vials and plates and then turn off your flame
14. Return all materials to where you got them and place your plates upside down in the 37°C Incubator
Note: Place any lids near your flame and upside down to prevent contamination
Note: Keep the cells in ice for as long as possible and only take them out when you are about to use them to streak and then immediately put them back.
2.2: Creating a liquid cell culture
Materials
Streaked plates
Streaking sticks
Sterile Falcon tubes
Pipette aid + Serological Pipettes
LB + Antibiotic Media
Instructions
Either wait 14-18 hours after streaking your plates in 2.1 to utilize those for the next part or utilize pre-streaked plates
Make sure your work area is aseptic
Retrieve two streaked plates (1 of pSB1-C3 on CHL and 1 of pECIA-K on Kan)
Place your streaked plates upside down on your bench and retrieve vials of LB + Antibiotic Media (In our case a vial of LB+CHL and LB+Kan)
Retrieve 4 Falcon tubes and label them with a permanent marker (your name, bench number, strain, plasmid). Label one tube as NEG Kan - this is your negative control. Two of the other tubes will be used for pSB1-C3 and the other one for pECIA-K
Turn on your flame before opening the vials of media
Using your pipette transfer 5mL of corresponding media into each culture tube (add 5mL of Kan to the pECIA-K and Neg Kan tubes and 5mL of CHL to the pSB1-C3 tubes). Ensure that any open vials or tubes are near the flame and their lids are placed upside down near the flame when open. Keep any containers open only for the minimum amount of time necessary.
Grab one of your plates and using a sterile streaking stick (run it through the flame first) dab an isolated colony. Place the plate back into its lid
Using your other hand, uncap one of the culture tubes and place your streaking stick into the tube.
Snap the top of the stick to ensure it fits in the falcon tube.
Cap your culture tube
Repeat steps 6-8 for the remaining tubes
For your negative culture tube place a sterilized streaking stick into it (no colonies)
Ensure that the lid of your falcon tube is only lightly screwed on (if you pull on the lid it should not come off but it shouldn't be fully tight) and then place a piece of tape over it to prevent it from falling off.
Turn off your flame and return all supplies to where you got them from
Place your culture tubes in a shaking 37°C Incubator overnight
Extra Resources
