Mar 13, 2026
F1-X™ Next-Generation 1-Step Gibson Assembly Protocol
- Racer Biosciences1,
- tei newman-lehman1
- 1Racer Biosciences
- Racer Biosciences
External link: https://www.racerbio.com/resources?tab=protocols-guides
Protocol Citation: Racer Biosciences, tei newman-lehman 2026. F1-X™ Next-Generation 1-Step Gibson Assembly Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9qob4l3e/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: March 12, 2026
Last Modified: March 13, 2026
Protocol Integer ID: 313194
Keywords: gibson assembly, dna cloning, seamless cloning, cloning, molecular biology, dna assembly, cloning kit, synthetic biology, f1-x, complete gibson assembly, f1, compatible with crude pcr product, crude pcr product, throughput workflow, racer bioscience
Disclaimer
For research use only. Not for use in diagnostic procedures. F1-X‱ is a trademark of Racer Biosciences. Gibson Assembly‱ is a registered trademark of Telesis Bio Inc., used under license. This protocol is provided for informational purposes only. Results may vary depending on DNA quality, fragment design, and laboratory conditions. Racer Biosciences assumes no responsibility for outcomes resulting from deviation from recommended conditions. For the most current version of this protocol, visit racerbio.com.
Abstract
A complete Gibson Assembly workflow using the F1-X‱ Master Mix from Racer Biosciences. F1-X‱ supports 2–12 fragment assemblies up to 100 kb in as little as 15 minutes. Compatible with crude PCR products and scalable down to 2.5 µL reactions for high-throughput workflows.
Guidelines
Keep F1-X‱ Master Mix on ice at all times. Do not store in a frost-free freezer. Limit freeze-thaw cycles to 5 or fewer — aliquot if using frequently. Always include three controls: positive assembly, vector-only (no insert), and no-assembly (no Master Mix). DNA quality is the most critical factor for success — always QC fragments before use.
Materials
All reagents required for the F1-X‱ assembly workflow are listed below. F1-X‱ Master Mix and Positive Control are available directly from Racer Biosciences at racerbio.com. Competent cells, nuclease-free water, and SOC medium are available from major life science distributors including Thermo Fisher and Sigma-Aldrich. For high-throughput workflows, F1-X‱ is verified down to 2.5 µL reaction volumes and is compatible with automation-friendly liquid handling systems.
Protocol materials
Reagent: F1-X™ Master Mix (2×)Racer BiosciencesCatalog #F1XGA10R
F1-X™ Positive Control (2×) Racer BiosciencesCatalog #F1XCTRL
Water, nuclease-freeThermo FisherCatalog #R0581
MAX Efficiency™ DH5α Competent CellsThermo FisherCatalog #18258012
SOC MediumMerck MilliporeSigma (Sigma-Aldrich)Catalog #S1797-10X5ML
Troubleshooting
Problem
No colonies from positive control
Solution
use high-efficiency cells (≥10⁹ CFU/µg), reduce freeze-thaw cycles, verify antibiotic plate freshness
Problem
No colonies from experimental samples
Solution
verify overlap length (40 bp recommended), column purify PCR products, check DNA quality and molar ratios using Qubit
Problem
High vector background
Solution
treat PCR vector with DpnI, gel purify to remove uncut plasmid
Problem
Low colony numbers
Solution
use fresh high-efficiency cells, optimize dilution factor, try electroporation
Problem
Inconsistent results
Solution
ensure reactions are assembled on ice, verify thermocycler temperature calibration
F1-X™ Assembly Protocol
1h 47m 30s
Fragment Preparation and QC. Verify concentration by Qubit fluorometer or NanoDrop spectrophotometer. Verify integrity by gel electrophoresis — greater than 80% full-length product required; gel extract if not met. Verify purity: A260/280 ≥1.8, A260/230 ≥2.0. Crude PCR products may be used at up to 20% v/v of reaction volume. Ensure homologous overlaps of 40 bp are designed into all fragment ends.
Equipment
Nanodrop™ One Spectrophotometer with WiFi and Qubit™ 4 Fluorometer
NAME
Spectrophotometer
TYPE
Thermo Scientific™
BRAND
13-400-525
SKU
LINK
Equipment
Qubit™ 3 Fluorometer
NAME
Fluorometer for nucleic acid quantitation
TYPE
Invitrogen
BRAND
Q33216
SKU
LINK
Equipment
Owl™ EasyCast™ B2 Mini Gel Electrophoresis Systems
NAME
electrophoresis system
TYPE
Thermo Scientific
BRAND
09-528-110B
SKU
LINK
Equipment
Microcentrifuge
NAME
ThermoScientific
BRAND
75002447
SKU
LINK
Calculate DNA Amounts and Prepare Fragment Mix. Simple assemblies (2–3 fragments): 0.03–0.2 pmols total DNA, vector 50–100 ng, vector to insert ratio 1:3 preferred. Complex assemblies (4–12 fragments): 0.2–0.8 pmols total DNA, 0.05 pmol per fragment target, equimolar ratio for all fragments. For fragments 100 bp or smaller, use 5× molar excess. Prepare DNA mixtures at greater than 2× final target concentration.
Set Up Assembly Reaction on Ice. Thaw F1-X™ Master Mix (2×) on ice. Vortex vigorously for 15 seconds before use. Combine on ice: 10 µL F1-X™ Master Mix (2×), X µL DNA fragment mix, nuclease-free water to bring total volume to 20 µL. Mix thoroughly and spin down briefly. For positive control: combine 10 µL F1-X™ Positive Control (2×) with 10 µL F1-X™ Master Mix (2×), no water needed.
Reagent: F1-X™ Master Mix (2×)Racer BiosciencesCatalog #F1XGA10R
F1-X™ Positive Control (2×) Racer BiosciencesCatalog #F1XCTRL
Water, nuclease-freeThermo FisherCatalog #R0581
0 °C
Equipment
Vortex Mixer
NAME
Vortex
TYPE
Fisher Scientific
BRAND
fisher scientific vortex mixer
SKU
Equipment
Microcentrifuge
NAME
ThermoScientific
BRAND
75002447
SKU
LINK
Incubate. For simple assemblies (2–3 fragments): incubate at 50°C for 15 minutes. For complex assemblies (4–12 fragments): incubate at 50°C for 60 minutes. For difficult assemblies, temperature may be raised to 53–56°C with incubation extended to 1–2 hours. Store completed reactions at −20°C or use immediately.
50 °C
00:15:00 60 minutes for complex assemblies
Equipment
T100 thermocycler
NAME
BioRad
BRAND
1861096
SKU
15m
Transform into Competent Cells. Add 1–2 µL of assembly reaction to 20 µL chemically competent cells on ice. Mix gently — do not vortex. Incubate on ice for 30 minutes. Heat shock at 42°C for 30 seconds. Return to ice for 2 minutes. Add 200 µL SOC medium. Incubate at 37°C for 1 hour with shaking. Plate on selective LB agar plates.
MAX Efficiency™ DH5α Competent CellsThermo FisherCatalog #18258012
SOC MediumMerck MilliporeSigma (Sigma-Aldrich)Catalog #S1797-10X5ML
4 °C
00:30:00
42 °C
00:00:30
4 °C
00:02:00
37 °C
01:00:00
1h 32m 30s
Colony Screening and Verification. Incubate plates overnight at 37°C. Screen 2–8 colonies for simple assemblies, 5–10 colonies for complex assemblies. Verify by colony PCR, miniprep and restriction digest, or Sanger sequencing. Sequence entire insert plus approximately 500 bp of vector at each junction. For full troubleshooting guidance download the F1-X™ User Guide at racerbio.com.
37 °C
Overnight
Protocol references
- Gibson DG et al. (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods 6, 343–345
- F1-X‱ User Guide v1.9, Racer Biosciences — racerbio.com
Acknowledgements
Protocol developed by Racer Biosciences, San Diego, CA. Based on the F1-X‱ User Guide v1.9. For technical support contact [email protected]. For purchasing contact [email protected].