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Protocol: Colony PCR
Preparation: Prepare a falcon tube for each colony you wish to test,
- Add 3ul of LB in each tube
- Add appropriate amount of the appropriate antibiotic
- Add 5 to 10ul of Lyse & GO (lysis reagent) into PCR tubes (one for each colony)
- Pick a colony on the plate
- Dip it in the falcon tube, don't drop the tip!
- Dip the same tip in the PCR tube with Lyse&GO.
- Repeat for each colony
- Put the PCR tubes in the PCR machine and select the Lyse&GO programm (10min at 95°C)
- Incubate the falcon tubes at 37°C.
PCR:
Chose an appropriate primer set to test your colonies, for example a forward primer specific to the backbone you want and a reverse primer specific to your gene of interest.
Volumes for a 20ul reaction, add in this order. (Multiply by the number of reaction you want to do +1)
| Reagent | Volume [μl] |
|---|---|
| Water | 20-x |
| HF-Buffer (5x) | 4 |
| DMSO (optional) | 0.6 |
| dNTPs | 0.4 |
| Forward primer (50μM) | 0.2 |
| Reverse primer (50μM) | 0.2 |
| Phusion HF polymerase | 0.2 |
Add 19ul of master mix to each tube and then add 1ul of colony lysate in the right tube (keep track of which colony is numbered how and in which tube!).
Controls controls controls!! Don't forget to have a positive control (for example, amplify your gene of interest from its original plasmid, this will directly give you the approximate size of the bands you expect), negative controls for your primer set and if colonies grew on you control plates test them with the same primer set as your test colonies.
