Plating Dictyostelium in soft agar

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[MATERIALS] - [INDEX]

Procedure

Note: These plates are not solid! DO NOT TIP.

1. Melt top agar solution and cool to 30°C.
2. 
   
3. Harvest Dicty cells from plate and pellet at 2K for 5 minutes.
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5. Aspirate media and suspend cells in 10 ml of HL-5 containing the appropriate drug concentration.
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7. Add drug to cooled top agar and add 40 ml to suspended cells.
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9. 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.
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11. Place plates on bench to solidify.
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13. Wrap up plates with moist towel and let sit for 10-14 days.
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15. To harvest a colony, suck the cells out of the agar with a pipetter.
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17. 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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