Transformation: Clonal Bacteria Picking Protocol

Materials Provided

• bacterial plates (our transformation plates)

• 1 cm transect squares

• Lauria Broth w/ 100µg/ml amp

Step 1

Wearing gloves, collect your bacterial plates

Step 2

Count the colonies on the plate. This may be complicated by the following factors:

• the presence of contaminant microorganism at the time of plating. Mold, fungus or unwanted bacteria can contaminate plates. If the plate contains furry or colorful colonies, it is indicative of contamination

• the presence of satelite colonies: small colonies which do not contain the resistance plasmid which can grow immediately next to larger colonies of bacteria, which do actually contain the plasmid

  • the bacteria containing the plasmid depletes the local concentration of ampicillin, allowing nearby bacteria to grow even in the absence of the resistance gene

• too many colonies: time consuming to count all

Shortcuts of Counting Colonies

• Count the colonies in ¼ or 1/8 of the plate and multiple to estimate the total number

• or use the 1 cm square cut outs to count colonies on 3 or 4 locations of the plate, average the number and extrapolate to the full surface of the plate (55cm²)

Step 3

From the count on the pUC transformation plate, calculate the transformation efficiency.

To do this, we need to know:

• how much pUC (mass) was put into the transformation?

• what fraction of the transformation volume did you plate?

• how many colonies did you obtain?

• transformation efficiency is the number of colonies/µg of pUC

Step 4

Provided you got colonies on your 1:1 or 1:3 pET32a-HA/gene ligation transformation plates, pick up to 3 colonies into separate round bottom tubes and add 2 ml of supplied liquid growth media

• label tubes and leave them in the provided rack

Step 5

• grow cultures overnight with shaking at 37ºC