Aspirate colonies with a spiking gun or Pasteur pipette and transfer to culture wells in culture flasks or multiwell plates.

Operation method

Scheme 14.8 Isolation of suspended cell clones

Principle

Aspirate colonies with a spiking gun or Pasteur pipette and transfer to culture wells in culture flasks or multiwell plates.

Materials and Instruments

Growth media Multi-well plates Culture flasks Anatomical microscopes Pipettes Bold-tip pens Yellow tips

Move

1. Observe the cells in the petri dish with an inverted microscope and mark the colonies with a felt-tipped stubby pen or Nikon marker.

2. Add 1 ml of medium to each well of a 24-well plate.

3. Pick the colonies under an autopsy microscope (20~50× magnification).

(a) Use one tip (yellow tip on the spiking gun) for each cell colony.

(b) Set the pipette to 100 μl.

(c) Start by aspirating approximately 50 μl of medium with the tip of the gun, aim this tip at the colony to be isolated and gently aspirate the remaining 50 μl within the colony.

4. Transfer the aspirate to a 24-well plate and rinse the colony with medium. If the medium contains Mexisol, the colony will fix, adhere, and grow; if the medium contains agar, the agar will be dispersed by blowing the colony up and down several times with a pipette in the culture well. The cell clones can also be transferred directly to 25 ^cm2 plastic culture flasks placed vertically.

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