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Creation and use of your infectious vector

Stewart Laboratory Standard Operating Procedure               


Soft Agar Assay

  1. Make 0.6% media-agar mix for the bottom layer.

        To make 0.6% agar mix the following components (this makes 200 ml):

  1. Prepare the top layer mixture with all of the listed components EXCEPT the Noble agar.

You will need one 50 ml conical for every two cell lines that you are testing. Prepare the following mix and place tube(s) at 37C to prewarm.

    Mix:

  1. 2X DME 12.5 ml
  2. IFS 2.5 ml
  3. Pen/Strep 0.25 ml
  4. ddH2O 3.5 ml
  5. Label tubes. For each cell line there should be 3-15 ml conical tubes labeled with 105, 104, or 103 and each of these should have the cell name on the tube.
  6. Lift two cell lines and pass through a 100μm cell strainer.
  7. Count cells.
  8. Resuspend the cells at 1 x 105/ml (make 5 ml).
  9. Set up dilutions as follows:
  10. Now melt the 2.4% Nobel agar that has been prepared in ddH2O (you made this earlier when you made the bottom layer).
  11. Add 6.2 ml Nobel agar to one of the 50 ml conical tubes and mix by inverting several times.
  12. Check to be sure that the contents of the tube are cool enough (you should be able to touch the inside of your wrist and it should be comfortably warm.
  13. Now you need to work fast but avoid air bubbles!!!!!!
  14. Add the following amounts of agar/media mix to each tube:
    1. Add 2 ml to the 103 and 105 tubes
    2. Add 7 ml to the 103 tube.
    3. Mix by inverting several times.
  15. Plate 4 ml of mix on each corresponding plate.
  16. Allow the plates to sit 30 to 60 minutes until they are solidified and then carefully transfer them to the incubator.
  17. Go back to step 4 for the next two cell lines.
  18. For long-term experiments you can feed the plates if they are looking dry or yellow with a 0.3% agar mix.