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scientificprotocols/protocol.md

Last active August 29, 2015 14:03
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Plating in Top Agar
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protocol.md
  1. Warm plates to room temperature before use. Cold plates causes the top agar to solidify irregularly. DO not warm plates to 37° as the top agar will take forever to solidify.
  • Prepare top agar as the appropriate liquid medium with 0.7% agar. Keeping 100 mL bottles is convenient. For phages, use λ top agar, which is less rich and yields bigger plaques.
  • Melt top agar in the microwave completely. Allow the agar to boil after liquification; incompletely melted agar looks liquid, but is grainy.
  • Allow agar to cool to 48°. Store in the 48° waterbath for convenience; old agar turns dark and should be discarded. Agar with drugs cannot be stored.
  • Add cells and phage to a 13 mm yellow-capped tube in the 48° temperature block.
  • Remove the cap and add 2.5 mL molten top agar. Do not replace cap.
  • Immediately vortex the mixture, flame the top, and dispense the mixture onto the appropriate plate. Swirl the plate to evenly distribute the agar.
  • Allow the plate to stand for several minutes as the agar cools.
  • After the top agar is solid, invert plate and incubate.

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