Colony Picking¶
Bench cheatsheet
Picking Colonies — the one-page version of this protocol. It is the same as the printed copies at the bench.
After plating your transformation and incubating overnight, if everything goes well you will see colonies that look something like this:
Figure: Results from two students' pP6 transformations under blue light illumination to excite amilGFP. Left: an ideal plate with many green colonies. Right: a minimally usable plate. The zoomed region shows a green colony (a true pP6 transformant) surrounded by smaller white "satellite colonies"—non-transformed cells growing in an antibiotic-depleted zone. Only the green colony contains the plasmid and is ampicillin-resistant.
What Are You Looking For?¶
In the pP6 experiment, you're trying to identify the strongest promoter variants. These drive expression of amilGFP, which fluoresces green.
- Bright green colonies indicate successful assembly of a strong promoter driving GFP.
- Colonies with no fluorescence are likely the template plasmid (pJ12) or inactive variants.
Some bright colonies are so strong that you can see yellowish color even without blue light. These are your best candidates.
Each visible spot on the plate arises from a single bacterium that landed there and divided repeatedly. To ensure you're picking a true single clone, choose well-isolated, round colonies that are clearly separated from their neighbors.
- Avoid picking colonies that are touching or very close together
- Avoid elongated or smeared colonies, which may indicate mixed or unhealthy growth
Picking refers to using a sterile tool (usually a toothpick or pipette tip) to transfer a single colony to a liquid culture.
Inoculating means introducing that colony into fresh media to grow overnight.
Figure: Use a sterile toothpick to gently touch a green colony, then inoculate 4 mL of media by dropping the toothpick into a labeled snap-cap tube.
Protocol¶
Follow your lab sheet and the steps below:
Step 1: Photograph the plate twice — under blue-light transillumination and under ambient light — and save both. Then mark the colonies you intend to pick.
Which colonies to choose is set by the experiment — brightest, smallest, largest, red, or deliberately naive. Your lab sheet says which; there is no general rule. Whatever the criterion, pick colonies that are easy to pick and look healthy: well separated, round and smooth. Big and well isolated is usually best. Avoid contorted or smudgy colonies — that appearance means water has seeped across the plate, or two colonies have grown into one.
Step 2: Label snap-cap tubes for up to 4 clones with your assigned ID number and a letter. For example:
pP6-79A, pP6-79B, etc.
Step 3: Add 4 mL of 2YT plus the antibiotic named on your lab sheet (from the fridge) into each tube. It is not always carbenicillin.
Tubes or a block. Up to 4 cultures, use individual snap-cap tubes as described here — each carries its own label, so the order you pick in does not matter. For more than that, use a 24-well block instead: its wells are identified by position, so fix the picking order before you start and write it down — use the grid on your lab sheet if it has one, and if it does not, draw the grid yourself and keep it with the block. Labels go on the airpore sheet rather than on the block.
Step 4: Pick each marked colony using a sterile toothpick and inoculate it by dropping the toothpick directly into the labeled tube (do not remove the toothpick).
Step 5: Cover the tube — caps on loosely, so the culture can breathe — and grow in the shaking incubator for 16–36 h. Refrigerate by 36 h; past that the cultures overgrow.
Step 6: Wrap the agar plate with parafilm and place it upside-down in the culture fridge for recordkeeping. The plate is a record, not a stock: the cells on it last 1–2 weeks even when wrapped. To keep a clone longer, restreak it or make a glycerol stock. See Plate Hygiene and Freshness.
Step 7: Upload a photo of your plate under blue light to this folder:
Google Drive Folder
Name the image as: pP6-79 (with your number)
Step 8: Document your picks. Why did you choose each clone?
(e.g., "very green and slow growing")
Example¶
| clone_id | why was it chosen? |
|---|---|
| 79A | It was exceptionally green and slow growing |
| 79B | Brightest colony on the plate |