Plating Dictyostelium in soft agar

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Procedure

1. Melt top agar solution and cool to 30°C.


2. Harvest Dicty cells from plate and pellet at 2K for 5 minutes.


3. Aspirate media and suspend cells in 10 ml of HL-5 containing the appropriate drug concentration.


4. Add drug to cooled top agar and add 40 ml to suspended cells.


5. Gently mix and pipet 5 ml of solution onto agar plates. It is best to pipet onto the plates in a swirling motion so that the solution is distributed evenly. DON'T try to swirl the media around on the plate by tilting it because the bottom agar will slide out.


6. Place plates on bench to solidify.


7. Wrap up plates with moist towel and let sit for 10-14 days.


8. To harvest a colony, suck the cells out of the agar with a pipetter.


9. After transfer to a plate they may take a few days to grow.

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Materials

• Top agar (40 ml per transformed plate)
  
  • HL-5 media
  • 0.38% low melting temp agar
  It is best to make up small (100 ml) aliquots
  
  
• Agar plates (10 plates needed per transformed plate)
  
  • HL-5 media
  • 0.3% Bactoagar
  • Appropriate drug (i.e. G418)

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