Silver: Colony picking using cloning rings

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Colony picking using cloning rings

There are several ways to obtain clonal populations after you stably transfect a cell line (using different approaches).

The way I do it involves using cloning rings (chemicon and others sell them w/ grease already, but you can just get regular cylinders and use your own grease instead) and it's very straight-forward. I'm using chemicon's TR-1004.

  1. After you have transfected and initiated selection for some days (depends on cell line efficiency, etc), take out the media
  2. Wash once with PBS and aspirate it
  3. Remove plate cover and tilt the plate towards you at an angle at which you can clearly see the surface of the plate with irregularities, such as, a new colony! This means you won't be picking colonies that are really really small. After you do it for a while, you'll get the hang of it. If you are doing the selection on small dishes such as 10cm or 6cm you can just put them under the microscope, look for colonies and mark them with a pen.
  4. Mark the colonies at the bottom of the plate using a pen, circular mark. Do not mark it exactly on top of the colony as it will make the cells harder to see on the microscope.
  5. Add PBS to the plate just to keep it "wet", not allowing the cells to dry
  6. Double check on the microscope that what you thought was a colony wasn't some sort of massive cell death or other debris. Celebrate if it's actually a colony that looks good!
  7. Repeat this for all plates/wells you have (celebrations included)
  8. Without removing the PBS off the plates, put the cloning rings on the plate surrounding the colonies, per your marks (they become useful now).
  9. Depending on how big the cylinders are, add 100 to 200 uL of trypsin (pre-warmed) and put it on the incubator, for 2-5min. Keep checking on them.
  10. I normally don't see the colonies "detach" in a similar fashion like one sees on a regular plate, but you certainly can see that they are kind of glowish on the membranes
  11. Add 100-200 uL of media (just to fill the cylinder) and pipette up and down to resuspend the cells
  12. Put them on a 24-well plate filled with 2mL of media

let them attach and expand a bit and watch their morphology on the microscope after some days.

Use all of your previous knowledge of the cell line you're working it when doing this process. If they colonies are really small and slow-growing, using even smaller wells than 24.

last edit bruno 14:58, 2 November 2006 (EST)

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