Team:DTU-Denmark/Methods/Colony PCR

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Revision as of 18:21, 2 July 2013

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:

1. water - 16 ul
2. Buffer - 2.5 ul
3. 5 mM dNTP mix - 1.25 ul
4. Primer 1 - 1.25 ul
5. Primer 2 - 1.25 ul
6. Polymerase - 0.25 ul
7. Cells - 2.5 ul (if suspended in water)or a small part of the colony

Total volume per one tube - 22.5 ul or 25 ul with suspended cells

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