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12.6: Exercise 2 - Replica plating and complementation

  • Page ID
    17571
  • This exercise will be performed at the next lab session after transformants will have had a chance to grow.

    Your initial selection of transformants was done on plates that lacked uracil, but contained methionine. Next, you will test the ability of your transformed strains to grow on media lacking methionine using replica plating. We will use methionine-free media containing either glucose or galactose for replicas, and you will also prepare a fresh master plate. Predict which transformants will grow on each of the plates.

    It is important to have a light touch during replica plating!! The goal is to transfer a small portion of cells from each colony on the master plate (the plates carrying your transfor- mants) to a number of plates containing different media.

    1. Place an orientation mark with a Sharpie on the perimeter of your master plate as well as the plates that will be used for replicas.

    2. Remove the lid from your master plate and invert the plate on the block, aligning the orienta- tion marker on the plate with the marker on the block. GENTLY and EVENLY tap on the bot- tom of the plate to transfer cells to the velveteen. Remove the master plate and replace the lid. You should see faint white colonies on the velveteen.

    3. Repeat step 3 with plates containing the following media:

    • Medium without uracil or methionine, containing glucose
    • Medium without uracil or methionine, containing galactose
    • Medium without uracil, containing glucose and methionine