Dec 22, 2025
Colony Screening of Agrobacterium
- Alexandra R. Grusky1,
- Christopher Dervinis2
- 1Department of Plant Molecular and Cellular Biology, University of Florida, Gainesville, FL, USA;
- 2School of Forest, Fisheries, and Geomatics Sciences, University of Florida, Gainesville, FL, USA
Protocol Citation: Alexandra R. Grusky, Christopher Dervinis 2025. Colony Screening of Agrobacterium. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gp1ekpgzp/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: December 19, 2025
Last Modified: December 22, 2025
Protocol Integer ID: 235485
Keywords: colony screening of agrobacterium, screening agrobacterium colony, agrobacterium colonies for the presence, throughput colony screening without genomic dna purification, agrobacterium, colony screening, throughput colony screening, direct pcr amplification, suitable for direct pcr amplification, genomic dna purification, using pcr, alkaline lysi
Abstract
This protocol provides a rapid and reliable method for screening Agrobacterium colonies for the presence of a construct of interest using PCR. Crude lysates generated by alkaline lysis are suitable for direct PCR amplification, allowing high-throughput colony screening without genomic DNA purification.
Guidelines
**Notes**
1. Buffer A must be prepared fresh for optimal lysis efficiency.
2. Avoid transferring excessive bacterial material, which may inhibit PCR.
3. Inclusion of PVP and BSA improves amplification by reducing PCR inhibitors.
Materials
**Reagents**
1. LB agar plates with appropriate antibiotics
2. Transformed Agrobacterium colonies
3. Buffer A: 100 mM NaOH, 2% (v/v) Tween 20 (prepare fresh)
4. Buffer B: 100 mM Tris-HCl, 2 mM EDTA (pH = 2.0; do not adjust)
5. Buffer C: 10% (w/v) PVP-40, 1% (w/v) BSA
6. PCR reagents and gene- or construct-specific primers
**Equipment**
1. PCR tubes (0.2 mL)
2. Micropipettes and sterile tips
3. Thermal cycler with heated lid
4. Vortex mixer (optional)
5. Agarose gel electrophoresis system
Troubleshooting
Colony Preparation (Timing: ~5 minutes)
Identify Agrobacterium colonies of interest on selective LB plates.
Replica plate or streak a small amount of each colony onto a labeled grid plate for identification and later recovery.
Cell Lysis (Timing: ~15 minutes)
Transfer a small amount of colony into a PCR tube containing 50 µL Buffer A. The bacterial mass should be just visible.
Repeat for all colonies to be screened.
Mix thoroughly by pipetting, vortexing briefly, or flicking the tubes to ensure bacteria are fully suspended. (Critical Step)
Incubate tubes at 95 °C for 10 minutes in a thermal cycler with the heated lid on. (Critical Step)
Neutralization (Timing: ~5 minutes)
Add 50 µL Buffer B to each tube to neutralize the lysate.
Mix thoroughly by pipetting, vortexing, or flicking the tubes.
PCR Amplification (Timing: 1.5–2.5 hours)
Prepare 25 µL PCR reactions according to the Taq polymerase manufacturer’s instructions, adding Buffer C at a 1:10 dilution and 1 µL lysate as template.
Run PCR using cycling conditions appropriate for the primers and target sequence.
Analysis (Timing: ~45 minutes)
Analyze PCR products by agarose gel electrophoresis.
Identify positive colonies and recover them from the replica or grid plate for downstream applications.
Protocol references
Xin Z, Velten JP, Oliver MJ, Burke JJ (2003). High-throughput DNA extraction method suitable for PCR. BioTechniques 34(4): 820–826.