Plate storage, use, and incubation

Plate Storage:
You will receive your plates at the same time you receive your competent cells. Plates should be stored at 4°C (in a regular refrigerator), upside down, until you are ready to use them. They can be removed from the refrigerator up to an hour before use.

Plate Use:
Be sure your students keep the plates closed except when they are plating the bacteria. Plates should only be opened using the “clamshell” or “umbrella” technique and only for as long as absolutely necessary.

Remind students to plate in order: (1) LB; (2) LB/Amp; (3) LB/Amp/Ara. This prevents movement of the ampicillin and arabinose between plates, which can lead to erroneous results. 

Once students have plated the bacteria, be sure the plates stay right side up for a few minutes to allow the bacteria to absorb into the agar, then place them upside down in the incubator (see below).

Incubating Plates:
Plates need to incubate at 37°C for at least 24 hours to ensure the growth of bacterial colonies. The abridged series has high enough transformation efficiency rates that if done properly, you should see pink colonies on the LB/Amp/Ara plate after 24 hours. The complete series can take longer, 36-48 hours, to see pink colonies and sometimes they are only visible with a UV or blue-light source. 

Please note that the more you open and close the incubator (to add plates from other classes, to check the plates, etc) the longer it will take your plates to grow colonies! We recommend adding all of a classes’ plates at one time, then keeping the incubator closed for a full 24 hours before you check for growth. If you have to open and close the incubator throughout the day to add plates from other classes, keep this to a minimum. Do NOT assume everything has failed if you do the labs one afternoon and don’t see growth the next morning. The plates generally need a FULL 24 hours or longer at a steady 37°C for the pink colonies to grow.
 

Resource category
ABE Lab Specific Resources
Related Lab Lab 5: Getting a Recombinant Plasmid into Bacteria Lab 5A: Getting a Recombinant Plasmid into Bacteria Lab 5B: Getting a Recombinant Plasmid into Bacteria