Team:Heidelberg/Notebook/Visualization/Methods

From 2008.igem.org

(Difference between revisions)
(→Swarm Assays)
(→Swarm Assays)
Line 487: Line 487:
*Step 3: Take Take a 50 ml falcon pour 25 ml of your agar-media in it and add antibiotics or inducing substances. ''If no extra substances are needed just skip this step.''
*Step 3: Take Take a 50 ml falcon pour 25 ml of your agar-media in it and add antibiotics or inducing substances. ''If no extra substances are needed just skip this step.''
*Step 4: Let your media polimerize. At least let it rest for 15/20 min.
*Step 4: Let your media polimerize. At least let it rest for 15/20 min.
-
*Step 5: Then add the attractant. e.g.: ''casamino acid plug''<ref>''casamino acid plug''<br>I used a solution containing 10% casamino acids and 2% agar called CA2A<br>Take a scalpel and cut off the upper part of a yellow pipette peak and put it upside down on your petri dish, there where you want the attractant plug to be<br>Heat the CA2A solution until it is fluid and the agar completely solved.<br>Put 100 µl of the CA2A solution inside the tube-top.<br>Let it rest for 10 minutes, then "gently" push the plug out of the tube-top, therefor you may use a blue pipette peak.<br>''There should still be a few samples of CA2A in the second -20 fridge, first shelf, blue rag, labeled GX. To get it fluid put it into the heating cube and after that a few times into the microwave, but just for 3-5 seconds, NOT at all longer!!! Kepp the tube away from you and open it gently after everytime you put it into the microwave.</ref>
+
*Step 5: Then add the attractant. e.g.: ''casamino acid plug''
*Step 6:
*Step 6:
**Put the whole plate into a 4°C fridge for 12-16 hours to allow the attractant to build up a gradient.
**Put the whole plate into a 4°C fridge for 12-16 hours to allow the attractant to build up a gradient.

Revision as of 19:10, 28 October 2008

Contents

Microscope

Small chamber

chemotactical activity

Large chamber

computer-assisted analysis

Swarm Assays

PROTOCOL FOR SWARM-PLATE CHEMOTAXIS AGAINST A POSSIBLE ATTRACTANT:
This protocol was created using petri-dishes. But every other chamber could be used as well.


  • Step 1: Make sure you have everything ready to get started. You need:
    • Petri-dishes. !!! sign your petri-dishes before You pour in the media. Also mark the spots where you want to set the attractant and the bacteria. !!!
    • 50 ml falcons
    • 25 ml media containig 0.2%-0.3% Agar for every petri-dish.
    • samples of the apropriate attractants.
    • Other substances like antibiotics (Amp, Kan, Cm) or inducer (IPTG, Arabinose)
  • Step 2: Your agar-media solution should be clear and the agar completely solved. If you want to use casamino acid plugs as attractant go first through step 5 then back through 2,3,4 skip 5,6 and so on...
  • Step 3: Take Take a 50 ml falcon pour 25 ml of your agar-media in it and add antibiotics or inducing substances. If no extra substances are needed just skip this step.
  • Step 4: Let your media polimerize. At least let it rest for 15/20 min.
  • Step 5: Then add the attractant. e.g.: casamino acid plug
  • Step 6:
    • Put the whole plate into a 4°C fridge for 12-16 hours to allow the attractant to build up a gradient.
    • Make an overnight culture of the strains you want to test, use TB media and add inducing substances if something has to be induced.

Next day:

  • Step 7: Take your overnight culture. It should be at an OD of 0.4-0.6. Then take 1 ml of the bacteria suspension and centrifuge it for 10 minutes at 4,000 rpm. Resolve the pellet at 100 µl of the media you are using in the petri-dish.
  • Step 8: Place 4 µl of your purrified bacteria at the premarked spot on the petri-dish sample.
  • Step 9: Put the whole plate into the incubator at ~30°C for 24 hours.


EXAMPLES:


Attractants: <references/>

Retrieved from "http://2008.igem.org/Team:Heidelberg/Notebook/Visualization/Methods"