Team:DTU-Denmark/Methods/Colony PCR


Colony PCR

Colony PCR

Colony PCR is a standard PCR with whole cells used as a template for verification purposes, for example to check if transformed cells contain the desired insert. Primers flanking insert are used.

Cells can be scraped directly from agar plates (using e.g. sterile tips or sterile wooden stick) and placed directly into the reaction mix or they can be suspended in water and 2.5 ul of it can be transferred to the reaction mix. Try to take a half of the colony by sterile tip, transfer it to the PCR mix in PCR tube and transfer the other half of the colony for a new plate (use flame!), which will contain all transformants choosen to be checked by colony PCR. Let the colonies transferred on a new plate grow during time and in the temperature optimal to your organism and after that keep it in the fridge until the result of colony PCR is known. If colony PCR will show positive colonies you can pick the positive transformant from this plate, inoculate liquid medium and save the successful transformant with glycerol at -80 °C.

Prepare a reaction mixture, or master mix if you plan to check many colonies, according to the procedure:

1x recipe for a 25uL PCR reaction

  • 5uL HF buffer
    • HF buffer should be 5x or adjust volume so final conc. of HF is 1x.
  • 0.5uL dNTP's
    • This can vary depending on conc. the final conc. should be 0.2mM.
  • 1.5uL of forward primer
  • 1.5uL fo reverse primer
    • The primer volume can differ quite a lot depending on which conc. you have them in. The final conc. of the each primer should be in the range of 1uM to 0.1uM but this can also be hard to generalize.
  • 0.25uL of polymerase
    • This again depend on the unit per microliter conc. of the polymerase. Use approx. 1.75U per reaction.
  • Cells (resuspend one colony in the MQ-water added to the reaction vial)
    • Take one colony, dissolve in 100uL milliQ and take 1uL from that as template.
    • The conc. of template can vary and this can also be one of the variables to turn on if the PCR goes wrong.
  • 15.75uL MQ-water
    • If using more or less of the previous reactant then just adjust the MQ amount so that the final volume will be 50uL

It can be a good idea to start labeling, then adding the MQ-water and then add cells by taking one colony and resuspending this in the water. Remember to save a little of the colony a plate it so that the colony don't get lots. When MQ and cells have been added all the rest of the components can be added as a master mix.

Now place PCR tubes in a PCR machine and use a standard PCR program or adjust parameters to the specific product and primers.