Colonies were digested with trypsin in porcelain cloning rings, glass rings, PTFE rings, or stainless steel rings and transferred to one well of a 24-well or 12-well plate or directly planted in 25 cm2 culture flasks.
Operation method
Scheme 14.6 Isolation of cell clones by cloning rings
Principle
Colonies were digested with trypsin in porcelain cloning rings, glass rings, PTFE rings, or stainless steel rings and transferred to a well in a 24- or 12-well plate or directly planted in 25 cm2 culture flasks.
Materials and Instruments
Trypsin Move 1. Observe the cell clones and mark the colonies to be isolated on the bottom of the petri dish with a felt-tip marker or Nikon marker (felt-tip marker or preferably Nikon marker or objective marker, which can be inserted into the position of the microscope's objective changer dial, to mark the selected clonal colonies). Caveat Petri dishes can dry out if exposed for too long, so limit the number of clones isolated or cover the dish between each step. For more product details, please visit Aladdin Scientific website.
Silicone oil
Cloning rings Pasteur pipettes Growth media Multi-well plates Sterile tweezers Pipettes Rough tip pens
2. Remove the medium and gently rinse the cell clones with D-PBS.
3. Pick up a cloning ring with sterile tweezers (dry heat or autoclaved in a Petri dish), dip it in silicone oil (dry heat sterilized in a glass Petri dish at 160°C for 1 h, or autoclaved at 121°C for 15 min), and place it on the Petri dish, silicone-side down, so that the silicone oil will be evenly distributed on the bottom surface of the cloning ring.
4. Put the ring on the desired colony.
5. Repeat steps 4 and 5 on the same petri dish, and attach 2~3 other desired colonies.
6. Add a sufficient amount of 0.25 % trypsin (0.25 % trypsin prepared in D-PBS) to fill the ring (0.1~0.4 ml, depending on the size of the inner diameter of the ring).
7. Retain for 20 s and discard.
8. Cover the petri dish and incubate at 37°C for 15 min.
9. Add 0.1-0.4 ml of medium to each ring.
10. Disperse the cells by pipetting for each clone in turn and transfer the cell suspension into a well of a 24-well plate or into a 25 cm2 culture flask placed vertically, using separate pipettes or lance tips (yellow lance or elbow pipettes and pasteurized pipettes with blunt tips) for each cell clone.
11. Rinse the cloning ring with 0.1-0.4 ml of culture medium and transfer the medium into the appropriate wells or flasks.
12. Add up to 1 ml of medium to the culture wells, cover and continue incubation. If culture bottles are used, add 1 ml of medium to each bottle and incubate vertically.
13. When the cloned cells have grown to the full size of the culture wells, transfer them to 25 cm2 culture flasks, add 5 ml of medium, and incubate them routinely. If the upright flask method is used, when the bottom of the flask is full of cells, remove the medium, trypsin digest, add 5 ml of medium, and place the flask flat for further incubation. 