Jun 25, 2025
Unmarked_Gene_Deletion_Ralstonia_Colony_PCR
- 1Lowe-Power Lab, UC Davis
Protocol Citation: Noah Guillome 2025. Unmarked_Gene_Deletion_Ralstonia_Colony_PCR. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldnojnv5b/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: June 24, 2025
Last Modified: June 25, 2025
Protocol Integer ID: 220959
Keywords: Cloning, Unmarked Deletion, Ralstonia, pUFR80, Targeted Gene Knockout, Gene Deletion, Homologous Recombination, Genome Editing, Chromosomal Integration, Gel Electrophoresis, DNA Gel Imaging, Colony PCR, Plasmid Screening, unmarked-gene-deletion-ralstonia-colony-pcr this protocol, unmarked-gene-deletion-ralstonia-colony-pcr, unmarked gene deletion in ralstonia, colony pcr screening step, gibson assembly for unmarked gene deletion, plasmid dna, unmarked gene deletion, transformation into ralstonia, pufr80 plasmid, experimental plasmid, plasmid, pcr product, execution of bulk pcr, bulk pcr, gene, pcr, ralstonia, dna, resistant to kanamycin, colony pcr
Abstract
This protocol describes the colony PCR screening step used to validate E. coli transformants carrying the experimental plasmid assembled during Gibson Assembly for unmarked gene deletion in Ralstonia. It outlines the setup and execution of bulk PCR to identify colonies that acquired the plasmid and are resistant to kanamycin.
The expected outcome is a ~1150 bp PCR product for colonies containing the pUFR80 plasmid with upstream and downstream inserts. Plasmid DNA will be extracted from correctly sized colonies and sequenced for verification prior to transformation into Ralstonia.
Guidelines
This protocol is intended for generating an unmarked gene deletion in Ralstonia using the pUFR80 vector backbone. It assumes that Gibson Assembly and subsequent E. coli transformation were performed according to the Unmarked_Gene_Deletion_Ralstonia_Gibson_Assembly_&_Transformation protocol, with upstream and downstream inserts cloned between the M13 primer binding sites. If plasmid assembly was carried out using a different strategy, the M13 primers may not be suitable, and modifications to this protocol may be necessary.
Materials
Reagents
- Isolated colonies from transformation plates (Unmarked_Gene_Deletion_Ralstonia_Gibson_Assembly_&_Transformation)
- pUFR80 plasmid DNA (undigested stock, for negative control)
- Autoclaved Milli-Q water
- Nuclease-free water
- M13 forward and reverse primers specific to pUFR80 backbone
- OneTaq‱ Hot Start Quick-Load‱ 2X Master Mix with GC Buffer (NEB #M0488)
- 1 kb Plus DNA Ladder (NEB #N3200)
- Loading dye (6X)
- Agarose powder
- Ethidium bromide (EtBr)
- Lithium Borate (LB) Buffer
- LB broth
- Kanamycin (25 mg/mL stock)
- 75% Glycerol (for backup stocks)
Equipment
- PCR tubes
- Microcentrifuge tubes (1.7 mL)
- Pipettes (P2, P20, P200, P1000) with sterile filtered tips
- Ice bucket and ice
- Microcentrifuge (for quick spin-downs and pelleting)
- Thermocycler
- Gel casting tray and comb
- Gel electrophoresis apparatus (gel box and power supply)
- UV gel imagining system (gel documentation system)
- Flash drive or data storage device
- Benchtop shaker incubator (37ºC, 250 rpm)
- Parafilm
- Waste container for Ethidium bromide gels and contaminated materials
Safety warnings
- Ethidium Bromide Safety: Ethidium bromide is a potent mutagen. Always handle it at a dedicated EtBr work station while wearing nitrile gloves. Avoid touching anything outside the station once gloved. All tips, gloves, gels, and materials that have contacted EtBr must be disposed of in the designated hazardous waste bins at the station. When loading PCR products onto the gel, dispose of one glove after pipetting, and replace it with a clean glove before handling any PCR tubes or equipment outside the EtBr zone. This minimizes contamination risk to samples, surfaces, and shared equipment.
- PCR Tube Sealing: Ensure PCR tubes are tightly sealed before placing them in the thermocycler. Improper sealing can lead to evaporation during high-temperature cycles, which may reduce yield or prevent amplification altogether.
- Gel Ladder Visibility: The smaller bands in the 1 kb Plus DNA ladder may appear faint and tightly spaced. For size estimation, prioritize the stronger reference bands at 0.5 kb, 1 kb, and 3 kb, which are more distinct and easier to identify.
- Troubleshooting Failed Screens: If no samples amplify to the correct size or all plasmid isolates have been sequenced and a correct clone has not been obtained, it is necessary to return to the E. coli transformation step in the Unmarked_Gene_Deletion_Ralstonia_Gibson_Assembly_&_Transformation protocol and screen a new set of colonies.
Before start
Follow Gibson Assembly and E. coli transformation steps in Unmarked_Gene_Deletion_Ralstonia_Gibson_Assembly_&_Transformation to obtain the 10 sample colonies that will be used for colony PCR.
Colony PCR
Sample Preparation
Label 10 PCR tubes for your colony samples.
Add 9 µL of autoclaved Milli-Q water to each tube.
Using a sterile pipette tip, pick an isolated colony from each of the 10 transformation plates and mix thoroughly into the respective PCR tube to disperse cells.
Spin down the PCR tubes.
PCR Setup
Prepare a PCR master mix on ice for 11 reactions (10 samples and 1 control), with an extra 10% to account for pipetting error, by adding the following in order. Mix by pipetting:
| Item | Volume per Reaction | Total Volume | |
| Autoclaved Milli-Q water | 3.6 µl | 43.56 µl | |
| OneTaq® Hot Start Quick-Load® 2X Master Mix with GC Buffer (NEB #M0488) | 5 µl | 60.5 µl | |
| M13F primer | 0.2 µl | 2.42 µl | |
| M13R primer | 0.2 µl | 2.42 µl |
Spin down the master mix.
Prepare PCR tubes:
- Aliquot 9 µL of the master mix into each PCR tube.
- Add 1 µL of each colony lysate to the corresponding sample tube.
- For the control tube, add 1 µl of empty pUFR80 plasmid to the PCR tube.
- Briefly spin down the PCR tubes.
Thermocycler Program
Briefly spin down both PCR tubes and load them into the thermocycler.
Run the following three-step PCR program (total reaction volume: 10 µL, lid temperature: 105ºC):
A. Initial denaturation: 94ºC for 30 seconds
B. 35 cycles of:
- Denaturation: 94ºC for 30 seconds
- Annealing: 53ºC for 30 seconds
- Extension: 68ºC for 1 minute 15 seconds (polymerase works at 15-30 seconds per kb)
C. Final extension: 68ºC for 5 minutes
D. Hold: 8ºC indefinitely
Start the program. Proceed to the next step during the run, or pause after completion and store PCR products at –20ºC.
Gel Electrophoresis
Gel Preparation:
- Make a 0.8% agarose gel in Lithium Borate buffer.
- Melt the agarose completely using a microwave.
- Add 0.2–0.5 µg/mL Ethidium bromide (1 drop per 50 mL).
- Pour gel into the casting tray with a comb inserted. Let it solidify for ≥30 minutes.
Sample Loading:
- Place the gel in the electrophoresis chamber and submerge it in Lithium Borate buffer.
- Load the wells:
- Well 1: 2 µL of 1 kb Plus DNA ladder (NEB #N3200)
- Well 2-11: 5 µL of sample PCR product mixed with 1 µL loading dye
- Well 12: 5 µL of empty pUFR80 PCR product mixed with 1 µL loading dye
Electrophoresis Setup:
- Seal the lid of the gel box.
- Connect the negative (black) lead of the power supply to the well side of the gel box and the positive (red) lead of the power supply to the far side.
- Set the power supply to run at 90 V for 30 minutes and press start.
Store the remaining PCR products at –20°C.
Product Validation
Gel Imaging
- Carefully remove the gel and place it in secondary containment.
- Image the gel using a gel documentation system.
- Save the gel image to a flash drive.
- Clean the gel station and dispose of the Ethidium bromide gel in the appropriate waste container.
Data Analysis:
- Compare band sizes to the ladder and no insert pUFR80 control.
- Expected result is for bands to be ~1150 bp if the sample contains the upstream and downstream fragments inserted into the pUFR80 plasmid.
- If samples contain empty pUFR80 plasmid, such as the no insert control, the PCR will only amplify a 146 bp region of the plasmid.
1 kb Plus DNA ladder (NEB #N3200) reference ladder
Liquid Culture Preparation
For colonies showing correct band size:
A. Prepare test tubes with:
- 3 mL LB broth
- 3 µL of 25 mg/mL kanamycin
B. Inoculate each with a colony from successful PCR reactions.
C. Incubate overnight at 37°C, shaking at 250 rpm.
Miniprep & Glycerol Stock
Use 500 µL of each overnight culture to make glycerol stocks (500 µL culture + 500 µL 75% glycerol).
Use the IBI Scientific I-Blue Mini Plasmid Kit (IB47170) to isolate plasmid DNA per manufacturer’s instructions.
Elute DNA in nuclease-free water.
Sequencing
Submit 10 µL of purified plasmid DNA (20–200 ng/µL) to a sequencing provider such as Plasmidsaurus (Oxford Nanopore sequencing).
Compare the sequence to the NEBuilder predicted construct in Benchling from the Unmarked_Gene_Deletion_Ralstonia_Primer_Design step.
If incorrect, sequence another plasmid isolate until a correct clone is identified or all samples have been exhausted.