Colony PCR

If starting from a sample from the -80° freezer:

1. Warm up appropriate plate. For S. pneumoniae, that means a blood plate. For E. coli, an LB plate with an appropriate antibiotic.
2. Take sample out of -80°C and place into a lab cooler from the -20° freezer. It is important that the sample does not thaw throughout this process. To help ensure that strains don't thaw, consider minimizing the number of strains out of the freezer by working with a smaller number of tubes at a time.
3. Important!: Complete the following steps in the hood if you are working with pathogenic bacteria.
4. Using a plastic inoculating loop, scrape a little piece of the frozen sample and streak it onto the plate.
5. Repeat step 4 for each sample you wish to grow up, using a new inoculating loop each time. Dispose of the used inoculating loop safely.
6. Incubate your plate.

After you have grown out your sample on plates:

1. Turn on one of the PCR machines. Set to incubate at 98° for an infinite time, and start the program.
2. Touch a P10 pipette tip to the outer edges of a colony. You are not pipetting the sample out; you are simply collecting the cells on a pipette tip.
3. Swish the pipette tip into a PCR tube filled with 50-200ul MilliQ water.
4. Heat at 98° for 10 minutes in the PCR machine. This will lyse the cells open so that DNA is exposed for PCR.
5. Use 1ul of the sample you just made as your template as per the Generic PCR protocol.