Colony PCR
Quick screen for whether a transformant carries the right insert. Pick a colony, lyse in the PCR, and read out a band on a gel within ~90 min.
Reaction setup (15–25 µL with Taq / OneTaq)
| Component | Volume (15 µL) |
|---|---|
| 2× OneTaq master mix | 7.5 µL |
| 10 µM forward primer | 0.3 µL |
| 10 µM reverse primer | 0.3 µL |
| Nuclease-free H₂O | to 15 µL |
| Colony | added by tip |
Use Taq, OneTaq, or any inexpensive polymerase — fidelity isn't important here. Don't waste Q5 on screening.
Procedure
- Number a fresh LB+antibiotic patch plate with a grid corresponding to the colonies you'll screen.
- Aliquot master mix into PCR strips on ice.
- For each colony: touch a sterile P10 tip to the colony, streak it onto the patch plate, then dip the tip into the master mix and swirl. Use minimal cells — large clumps inhibit PCR.
- Incubate the patch plate at 37 °C overnight; use this for downstream miniprep of confirmed clones.
- Run thermocycler:
- 95 °C, 5 min (lyses cells)
- 30 cycles: 95 °C 30 s · Ta 30 s · 68 °C 1 min/kb
- 68 °C, 5 min · 4 °C ∞
- Run 5 µL on a 1% agarose gel. See agarose gel.
Tips
- Always include a positive control (e.g., parent plasmid) and an NTC.
- For low-abundance plasmids or genome integrations: use 95 °C / 10 min initial lysis.
- If you only get smears, you've added too many cells. Touch the colony lightly.
- Confirm 1–2 positive bands by Sanger or whole-plasmid sequencing — colony PCR can give false positives from primer dimers or cross-priming.