Lab Procedures
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Nic Theobald Yilectronics
Introduction:

Congratulations! You've made it! Hopefully, by now, you are at least familiar with the previous topics (if not, I'll also have a link to the corresponding topic). Now, we can actually go over the procedures to recreate our results.

First, we will

Creating Cuture Media:
In order to preserve an initial culture of cells you must create a new one that you can actually use in your experiments. That and your initial culture may not have individual colonies. We will start by creating the actual plate and liquid medium. Before starting, ensure that all tools and equipment that touch post-autoclaved materials are sterile. Also, ensure that your workspace is clutter free and clean.

Plate Media (read directions on container, adjust quntities to suit need)
  1. Add 35 grams of "LB Agar" (liquid broth agar) and 1L of distilled water to a threaded jar.
  2. Add stir bar and heat until all contents are completely dissolved.
  3. Leave stir bar in the jar and and thread on the lid so it wont fall off if bumped. Do not tighten, gas should be able to vent from the jar!
  4. Label the jar with a strip of heat sensitive tape.
  5. Place in a container so that boiled over contents don't get into the autoclave.
  6. Place jar (stir bar included) into the autoclave.
  7. Ensure autoclave has enough water.
  8. Start the autoclave by pressing the "Liquids" button.
  9. Leave in the autoclave for 15 minutes.
  10. Remove the jar from the autoclave using heat protective mits and set on the counter.
  11. While the agar is still hot, pour into sterile petri dish.
  12. Place the agar dish on its lid in the refrigerator until the media has solidified.
  13. Remove the stirbar and cleanOnce the media has solidified, draw these quadrants on the bottom the the plate media.
  14. There are two options, choose whichever suits your needs.

Liquid Media (read directions on container, adjust quntities to suit need)
  1. Add 20 grams of "LB Broth" (liquid broth) and 1L of distilled water to a threaded jar.
  2. Add stir bar and heat until all contents are completely dissolved.
  3. Leave stir bar in the jar and and thread on the lid so it wont fall off if bumped. Do not tighten, gas should be able to vent from the jar!
  4. Label the jar with a strip of heat sensitive tape.
  5. Place in a container so that boiled over contents don't get into the autoclave.
  6. Place jar (stir bar included) into the autoclave.
  7. Ensure autoclave has enough water.
  8. Start the autoclave by pressing the "Liquids" button.
  9. Leave in the autoclave for 15 minutes.
  10. Remove the jar from the autoclave using heat protective mits and set on the counter.
  11. Transfer the liquid media to a sterile test tube and label with a piece of tape.
  12. Place the test tube in a test tube rack for later use.
Innoclating Liquid Media
  1. Prepare bunsen burner for innoculation loop flaming.
  2. Ignite the bunsen burner.
  3. Loosen the cap of the test tube that contains the liquid media.
  4. Prepare your initial cell culture for transfer.
    1. If your initial plate culture has individual colonies, circle the colony you are taking cells from.
    2. If your initial plate culture is one large colony, mark where you you are taking cells from.
    3. If you are taking cells from a liquid culture, don't worry about which "colony" you are taking from.
  5. Flame the innoculation loop and wait a moment for it to cool
  6. Perform the next two steps rapidly in order to reduce the amount of time the cultures are exposed to open air.
    1. For taking cells from a plate culture, quickly remove the lid of the dish and touch th.e end of a sterilized innoculation loop to one of the colonies.
    1. For taking cells from a liquid culture, stir the end of a sterile innoculation loop in the liquid culture.
  7. Replace the lid or cap on the dish or tube and remove the cap of your uncultured liquid media.
  8. Stir the innoculation loop in the media and quickly replace the cap.
  9. Label the test tube and set aside for incubation.

Innoclating Plate Media
  1. Prepare bunsen burner for innoculation loop flaming.
  2. Ignite the bunsen burner.
  3. Prepare your initial cell culture for transfer.
    1. If your initial plate culture has individual colonies, circle the colony you are taking cells from.
    2. If your initial plate culture is one large colony, mark where you you are taking cells from.
    3. If you are taking cells from a liquid culture, don't worry about which "colony" you are taking from.
  4. Flame the innoculation loop and wait a moment for it to cool
  5. Push the end of the innoculation loop into the edge of the uncultured plate media until it stops hissing.
  6. Perform the next two steps rapidly in order to reduce the amount of time the cultures are exposed to open air.
    1. For taking cells from a plate culture, quickly remove the lid of the dish and touch th.e end of a sterilized innoculation loop to one of the colonies.
    1. For taking cells from a liquid culture, stir the end of a sterile innoculation loop in the liquid culture.
  7. Replace the lid or cap on the dish or tube and remove the cap of your uncultured plate media.
  8. Touch the end of the innoculation loop to the plate media furthest away from the next quadrant.
  9. Completly fill in the area by zigzagging the loop across the first quadrant. Try to fill the entire area, but DO NOT backtrack to fill in empty spaces and DO NOT pick up the loop midway through.
  10. Flame the innoculation loop and wait for it to cool.
  11. Then touch and drag from the first quadrant into the second quadrant and repeate the process for the rest of the quadrants.
  12. Set the innoculated plate media aside for incubation.
The whole point is to create a concentration gradient of cells from quadrant 1 to quadrant 4. Once incubated, the first quadrant should be a large mass of cell growth, the second should have a lesser mass and a few colonies, the third should be all colony, and the fourth should have some colonies and fade to nothing.


Incubating
  1. For plate cultures, place the cultures upside down in the incubator.
    1. You can stack them if you like.
  2. For liquid cultures, place them in the test tube shaker.
    1. Set the shaker to its midway speed.
  3. The incubator should already be turned on a set to ___.
  4. Close the door of the incubator and let them sit for 24-48 hours.
  5. Check them at after 24 hours to guage when to take them out.
    1. You dont want the colonies to migrate far beyond their original location.


Performing PCR
Finally, we can move on to the whole purpose of this lab.


Procedures for Freezing:
Creating 45% Glycol Mixture

  • Slowly pipette 450 microlitres of glycerol into a small test tube (it may take fractions of a minute just to draw the glycerol into the pipette)
  • Pipette 550 microlitres of deionized water into the same small test tube
  • Agitate the test tube until the viscous glycerol is completely mixed with the water
Preparing Cryovials
  • Slowly pipette 1 milliliter of the Glycol mixture and 2 milliliters of the liquid culture into the cryovials
  • Label the cryovials
  • Put on low temperature safety mits
  • Take the top off the liquid nitrogen vessel and carefully collect a small amount into a low temp safe container
  • Drop the cryovials into the container without splashing any of the liquid nitrogen
  • Remove the vials with temp safe tongs and place in -80C freezer




Page created by Nic Theobald 8/9/2020
Last Update: 8/27/2020