Oct 05, 2025
- igemnthuth 1
- 1NTHU_igem
- iGEM NTHU
Protocol Citation: igemnthuth 2025. Colony PCR. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpm5x8gzp/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 03, 2025
Last Modified: October 05, 2025
Protocol Integer ID: 228961
Keywords: colony pcr, target dna fragment, colonies by pcr, pcr, dna, transformed colony, colony
Abstract
Rapidly screen transformed colonies by PCR to determine whether they successfully contain the target DNA fragment.
Materials
(I) Colony Source
- Single colony grown 16–18 hr on an LB agar plate containing the appropriate antibiotic
(II) PCR Reagents
- Forward and reverse primers
- 10× Taq polymerase buffer
- ddH₂O
- Taq DNA polymerase
- 10 mM dNTPs
(III) Instruments / Equipment
- Thermal cycler (PCR machine)
- PCR tubes (0.2 mL)
- Microcentrifuge tubes
- Micropipettes (pipetman)
- Microcentrifuge
- Vortex mixer (or shaker)
Troubleshooting
Colony Picking and Template Preparation
Select colonies from the agar plate. Inoculate a portion of each colony into LB medium containing the appropriate antibiotic and incubate at 37 °C. Place the remaining portion of the colony into a correspondingly numbered PCR tube containing 25 µL of reaction mix (see below), and pipette to fully resuspend the bacteria.
PCR Reaction Setup (20 µL per reaction)
Prepare the PCR reaction mix before starting Step 1 and keep the mix on ic
Gently mix the reaction components, then briefly centrifuge the tubes to collect the contents at the bottom.
Place the PCR tubes into the thermal cycler and run the following program: