User Guide

F1-X™ Next-Generation 1-Step Gibson Assembly® Master Mix

Revision 1.9 | Effective 03 March 2026 | © 2026 Racer Biosciences

Preface

Over the past two decades, molecular biology has been transformed. DNA sequencing has become faster and more accessible, and the cost of synthetic DNA continues to decline. As these technologies advance, the ability to assemble DNA efficiently, accurately, and at scale must evolve in parallel.

At Racer Biosciences, our mission is to create next-generation tools that accelerate innovation in DNA assembly and preparation. The F1-X™ Next-Generation 1-Step Gibson Assembly® Master Mix embodies that mission, bringing better performance, precision, and capabilities to a trusted workflow.

Over a decade ago, the invention of the Gibson Assembly method revolutionized cloning by enabling seamless, one-step assembly of multiple DNA fragments without the need for restriction enzymes or ligation scars. Since its introduction, the method has been cited in over 40,000 publications worldwide.

Building on the simplicity and legacy of the Gibson Assembly method, F1-X™ offers:

• Fast and simple assembly: complete reactions in a 1-Step process in as little as 15 minutes
• Efficient multi-fragment assembly: high-fidelity and effective assembly excels with complex builds
• Visual positive control: measure cloning efficiency quickly and easily
• Small volume and automation friendly: verified down to 2.5 µL, F1-X™ is optimized for high throughput and low waste workflows

Whether you are building expression vectors, genetic circuits, or entire pathways, F1-X™ delivers robust results and accelerates your innovation. This manual guides you through preparation, assembly, and transformation using F1-X™.

At Racer Bio, we prioritize our customer experience and quality above all else.

For help and product feedback, contact info@racerbio.com
For purchase questions, contact sales@racerbio.com

Table of Contents

Legal Notices

Limited Use Label License

The F1-X™ Next-Generation 1-Step Gibson Assembly® Master Mix, and components and products thereof, is to be used for internal research purposes for the sole benefit of the purchaser only. They may not be used for any other purpose, including, but not limited to, use in drugs, diagnostics, therapeutics, or in humans. The F1-X™ kit and components and products thereof may not be transferred or sold to third parties, resold, modified for resale, used to manufacture commercial products, or to provide a service of any kind to third parties, including, without limitation, reporting the results of purchaser's activities for a fee or other form of consideration. Except as otherwise agreed in writing by our authorized representative, this product is for internal research use only and not for human, animal, therapeutic, or diagnostic use.

Limited Warranty

The F1-X™ kit, and components and products thereof, is warranted to perform according to specifications stated on the certificate of analysis. No other warranty is made, whether express or implied, including any warranty of merchantability or fitness for a particular purpose. This warranty limits Racer Biosciences Inc.'s and its licensors' liability to only the price of the kit. Neither Racer Biosciences nor its licensors shall have any responsibility or liability for any special, incidental, indirect, or consequential loss or damage whatsoever.

Disclaimer

The material in this manual is for informational purposes only and is subject to change without prior notice at any time. Racer Bio and/or its affiliates assume no responsibility for any errors that may appear in this document.

Trademark Information

F1-X™ is a trademark of Racer Biosciences. Gibson Assembly® is a registered trademark of Telesis Bio Inc., used under license. This guide references third-party brands to identify products that are compatible with, or commonly used alongside, the F1-X™ Next-Generation 1-Step Gibson Assembly® Master Mix kit. All such names are the property of their respective owners, and their use herein does not imply endorsement or affiliation.

Patents

F1-X™ technology is protected under US patent numbers 7,776,532, 8,435,736, 8,968,999, and additional patents pending.

Regulatory Statement

For research use only. Not for use in diagnostic procedures.

Kit Information

F1-X™ Next-Generation 1-Step Gibson Assembly® Master Mix

Compatibility and Features

Principles of Gibson Assembly® Technology

The Gibson Assembly® method enables seamless, 1-Step joining of multiple DNA fragments without restriction enzymes or ligation scars. F1-X™ builds upon this original chemistry with enhanced speed, fidelity, and flexibility. The method relies on user-defined overlapping ends designed into fragments, allowing homologous overlaps to guide assembly. DNA fragments with homologous overlaps are combined with the Gibson Assembly® Master Mix and incubated at a constant temperature (50–56°C). Within this isothermal environment, three coordinated enzymatic activities work in concert (Figure 1).

Figure 1. Gibson Assembly Method Overview. Schematic showing the three enzymatic activities (exonuclease, polymerase, and ligase) working together to join DNA fragments with homologous overlaps in a single isothermal reaction. Note that overhang design dictates the circularity or linearity of the final assembly product. Reaction efficiency is influenced by DNA quality, design, concentration, and reaction time/temperature.

F1-X™ Workflow Overview

The F1-X™ workflow is engineered for simplicity, precision, and speed:

• Fragment preparation: Generate DNA fragments with homologous overlaps (typically 20–40 bp for simple assemblies or up to 80 bp for complex constructs). Fragments can be generated by PCR or restriction digestion, or sourced from DNA synthesis providers. Quality control, outlined in this manual, is an essential step of the process.
• Assembly: Combine mixture of fragments with F1-X™ Master Mix (2×) and incubate at 50°C for 15–60 minutes.
• Downstream applications: Use assembled DNA directly for transformation without purification. Alternatively, use the assembled construct as a template for traditional PCR or rolling circle amplification (Figure 2).

Figure 2. Workflow diagram. DNA fragments with homologous overlaps are assembled in 1 Step using F1-X™ Master Mix (2×). The resulting construct can be directly used downstream.

DNA Fragment Preparation and Quality Control

Essential Quality Metrics and Measurement Methods

All DNA fragments require assessment of these three parameters:

Concentration Measurement Methods

Integrity Measurement Methods

Common DNA Purification Methods

Assessing DNA Purity

Spectrophotometric Analysis:

• A260/A280 ratio: Assesses protein contamination. Pure DNA: 1.8–2.0
• A260/A230 ratio: Detects organic compounds and salts. Pure DNA: >2.0
• RNA contamination: Increases A260/A280 ratio >2.0 and inflates concentration estimates

Fragment Preparation Methods

PCR Amplification (Recommended)

PCR is the most versatile method for adding homologous overlaps to an existing piece of DNA. The overall workflow includes:

1. Design primers with correct binding region and homologous overlap region
2. Assemble PCR reactions using high-fidelity polymerase (Platinum™ SuperFi™ II [Thermo-Fisher] and Q5® [NEB] for example)
3. Digest residual template by adding DpnI; incubate 30 minutes–2 hours at 37°C
4. Analyze by electrophoresis and gel extract if low molecular weight contaminants are present
5. Purify if needed, or use directly with F1-X™

Recommended PCR Cycling Conditions

*Calculate based on polymerase vendor's Tm calculator

Restriction Enzyme Digestion

For vector linearization or fragment excision:

1. Digest with appropriate restriction enzyme(s) according to manufacturer's instructions
2. Treat with CIP (Calf Intestinal Phosphatase) to prevent self-ligation
3. Heat inactivate enzymes per manufacturer's recommendations
4. Gel purify linearized vector to remove uncut plasmid and contaminants
5. Quantify and assess purity before proceeding to assembly

Vector Preparation Workflows

Vectors for assembly can be prepared through multiple approaches, each with specific advantages depending on your source material and throughput requirements. Figure 3 shows a comparison of restriction enzyme digest and PCR options. Optional QC for vector includes transforming linear vector directly to cells to ensure no background colonies are present, and performing a vector-only control to ensure no self-ligation.

Figure 3. Vector Preparation Workflow Examples. Flowchart showing example workflows for vector preparation from plasmid DNA, including restriction enzyme digest or PCR amplification approaches.

Homologous Overlap Design Guidelines

General Design Principles

• Recommended starting point: 40 bp overlaps for all assemblies
• Minimum overlap: 20 bp (acceptable for simple 2–3 fragment assemblies)
• Complex assemblies: Use 40+ bp overlaps for optimal efficiency
• Avoid: Highly repetitive sequences, extremely low/high GC content
• Ensure: Overlaps are unique within the construct

Suggested Overlap Lengths by Fragment Size and Number

2–3 Fragment Assemblies

4–12 Fragment Assemblies

Vector-Specific Recommendations

Design Tools

Figure 4 shows a visual depiction of how overlaps for Gibson Assembly work. For complex constructs, consider using the DNA Fragment Splitter for Gibson Assembly tool, which provides automatic fragment splitting with optimized overlaps, synthesis-ready FASTA exports, built-in QC and annotations, and expert-guided feedback in the interface.

Figure 4. Example of a 40 bp homologous overlap (shown in CAPS) used to join two adjacent DNA fragments. All sequences are shown from 5' to 3'. The F1-X™ Master Mix enzyme blend chews back the 5' ends, allowing the complementary overlap regions to anneal, then extends and ligates to create a seamless junction.

F1-X™ Assembly Protocol

General Guidelines

• Keep F1-X™ Master Mix on ice at all times. Assemble reactions on ice.
• Include appropriate controls.
• Follow DNA QC procedures precisely.

Calculating DNA Amounts and Ratios

DNA mixtures should be prepared at >2× of the final target concentration. If scaling down, scale DNA amounts linearly with reaction volume.

Target Ratios and Amounts (final amounts in 20 μL reaction):

* For fragments ≤100 bp, use 5× molar excess.

Quick Reference Table

To determine the pmols or ngs of DNA:

Controls

• Positive: F1-X™ Positive Control (2×) or DNA from a previously verified assembly (see Appendix A)
• Negative: Vector only (no inserts) to assess background
• No-assembly: DNA fragments without F1-X™ Master Mix for gel analysis reference

Assembly Procedure

Materials: F1-X™ Master Mix (2×), F1-X™ Positive Control (2×), DNA fragments, nuclease-free water, thermocycler

Protocol:

1. Thaw F1-X™ Master Mix (2×) on ice
2. Prepare DNA fragments by mixing in nuclease-free water according to calculated ratios
3. Vortex Master Mix vigorously for 15 seconds before use
4. Combine on ice:

For Positive Control: add 10 µL F1-X™ Positive Control (2×) to 10 µL F1-X™ Master Mix (2×) for a 20 µL reaction.

5. Mix well and spin down briefly
6. Incubate and assemble:
   • Simple assemblies (2–3 fragments): 50°C for 15 minutes
   • Complex assemblies (4–12 fragments): 50°C for 60 minutes
7. Store at -20°C or use immediately for transformation/downstream applications
8. Optional post-assembly analysis: run 5–7 μL on an agarose gel to verify assembly. Include no-assembly control for reference, as assembled DNA may appear faint.

F1-X™ Transformation Protocols

Transformation Guidelines

Optimization of dilution factor and amount of cloning reaction to transform is essential for achieving robust colony counts. The suggestions below are guidelines for standard chemically and electrocompetent cell workflows.

Competent Cell Selection

Chemically Competent Transformation Protocol

Guidelines for using DH5α™, NEB® 5-alpha in 96-well plate format are shown below. Always refer to competent cell manufacturer guidelines for more details. If using 50 μL of competent cells in microcentrifuge tubes, transform 2.5 μL of the cloning reaction.

1. Add 20 μL competent cells to pre-chilled tubes or deep 96 well plate on ice
2. Add 1 μL of F1-X™ assembly reaction (neat) to 20 μL cells and mix gently on ice
3. Incubate on ice for 30 minutes
4. Heat shock at 42°C for 30 seconds
5. Return to ice for 2 minutes
6. Add 200 μL SOC medium; incubate 37°C for 1 hour (with shaking for high efficiency)
7. Plate cells using patch plate, streak, or beads/spreaders as needed. See Plating Guidelines section for more information

Electrocompetent Transformation Protocol

Guidelines for using TransforMax™ EPI300™ or equivalent. Always refer to competent cell manufacturer guidelines for details.

1. Dilute F1-X™ reaction 3–5 fold in ice-cold water
2. Add 2 μL diluted reaction to 30 μL cells on ice; mix gently
3. Transfer to pre-chilled electroporation cuvette
4. Electroporate: 1200V, 25μF, 200Ω (0.1 cm cuvette)
5. Immediately add 950 μL SOC to cells for recovery; incubate 37°C for 1 hour with shaking
6. Plate cells using patch plate, streak, or beads/spreaders as needed. See Plating Guidelines section for more information

Plating Guidelines

Traditional Plating

*Conservative estimates where percentage refers to % of culture rescue. †Colony counts are estimates and will vary based on assembly complexity, efficiency, DNA quality, and design. ‡Centrifuge briefly and remove supernatant before streaking the pellet.

Patch Plating Protocol for High-Throughput Workflows

Since the goal of transformation in cloning is typically to obtain individual colonies for sequencing, plating serial dilutions of transformation culture helps determine optimal plating volumes before sequencing while conserving reagents.

1. In a 96-well plate, dilute each rescue culture 1:10 and 1:100 in SOC
2. Using a multichannel pipette, gently patch 2.5–5 μL of each dilution onto selective agar plates, keeping droplets as individual patches
3. Incubate plates overnight at the appropriate temperature (typically 37°C) and store transformation cultures at 4°C
4. Identify conditions with individual colonies to calculate CFU per μL, then use optimal culture volume for sequencing plates. Note that transformation cultures in SOC can be stored at 4°C overnight while patch plates are grown

Colony Screening and Verification

Colonies can be pre-screened for full length assembly product via colony PCR, miniprep/digest, or screened directly from the agar plate depending on the desired workflow.

Colony Selection Guidelines (Full-Length Constructs to Screen)

Note: Requirements are sequence dependent. Factors include fragment sizes, fragment error rates, GC content, DNA quality, overlap length, and presence of toxic sequences. Pre-screening colonies with colony PCR or miniprep and restriction digest is recommended for complex assemblies.

Appendix A: Protocol and Expected Results from F1-X™ Positive Control (2×)

The F1-X™ Positive Control (2×) can be used to verify proper assembly conditions and transformation efficiency.

Positive Control Composition

The F1-X™ Positive Control (2×) contains a 4-fragment assembly designed to produce a functional GFP expression cassette in a pUC vector. The mixture includes three insert fragments of ~0.7 kb and one linearized pUC vector fragment (2.8 kb), present at approximately equimolar ratios (1:1:1:1). All fragments contain 40 bp homology regions for seamless assembly. Upon successful assembly, the fragments combine to form an approximately 4.7 kb circular plasmid containing an intact GFP gene under constitutive expression.

Protocol

1. Assembly Reaction Setup — Prepare a 20 µL reaction as follows:

2. Mix gently and incubate at 50°C for 15–60 minutes. Note that the positive control contains 4 fragments. While this is technically within guidance for a 60 minute reaction time, this particular assembly has been verified down to 15 minutes for ease of use alongside simple assemblies.

3. Transform according to guidelines above alongside experimental samples.

Expected Results

• Colony count: >100 colonies per plate
• Appearance: Green fluorescent colonies visible under ambient light
• Expected efficiency: Typically >90% under recommended conditions

Note: If fluorescence is difficult to visualize under room light, illuminate plates with blue light or use colony count as the primary success metric.

Figure 5. (a) Schematic of the 4-fragment assembly. Three insert fragments (1, 2, 3) and one linearized vector fragment (4) are assembled into a circular plasmid containing an intact GFP gene. (b) Agarose gel electrophoresis showing assembly products. Without Master Mix (left lane), distinct bands are visible for the vector (2.8 kb) and three insert fragments (~0.7 kb each). With Master Mix assembly at 50°C for 15 minutes (right lane), high molecular weight products are observed. (c) Transformation results showing green fluorescent colonies.

Appendix B: Seamless Removal of Restriction Enzyme Seams with the Gibson Assembly® Chemistry

Gibson Assembly chemistry can eliminate restriction enzyme seams during assembly. The 5′ exonuclease activity removes restriction site overhangs, while polymerase and ligase activities create seamless junctions between fragments with homologous overlaps.

Figure 6. Elimination of EcoRI Seam During Gibson Assembly. Diagram depicting two fragments with homologous overlap regions (shown in orange) and an external EcoRI site. Following restriction enzyme digestion with EcoRI, the fragments may be assembled using the Gibson Assembly reaction. The restriction enzyme digestion seam is eliminated by the 5' chew back and ligation activities of the F1-X™ Master Mix.

Troubleshooting Guide

Common Assembly Issues

Common DNA Preparation Issues

Optimization Strategies

For Difficult Assemblies

• Increase reaction temperature to 53–56°C for higher stringency and extend incubation time up to 1–2 hours
• Use longer overlaps (60–80 bp for complex assemblies)
• Consider two-stage assembly for >12 fragments
• Consider whether the competent cell strain being used is appropriate

For Low Efficiency

• Verify all fragments are full-length by gel
• Use higher DNA concentrations within recommended range
• Ensure proper molar ratios (equimolar for complex assemblies)
• Column purify all DNA fragments before assembly
• Column purify assembly reaction to desalt before transformation
• Consider electroporation vs. heat shock

For Repetitive Sequences

• Design overlaps to avoid repetitive regions
• Use longer overlaps to increase uniqueness
• Consider fragments with repetitive sequences internalized
• Perform sequential assembly stages

FAQ: General Questions

What are the advantages of Gibson Assembly®?
1-Step, seamless cloning without restriction site dependence; scarless junctions with no unwanted sequences; versatile for genes, plasmids, pathways, and whole genomes; direct use for transformation, PCR, or rolling circle amplification.

Can I amplify the assembled product directly?
Yes. The covalently joined DNA can be used as a PCR template or for rolling circle amplification without purification. As a starting point, we recommend testing 1 µL of F1-X™ reaction in a 20 µL PCR or rolling circle amplification reaction following manufacturer guidelines.

Can I combine ssDNA oligonucleotides with dsDNA fragments?
Yes. Use 45 nM as starting concentration for each oligonucleotide. Oligonucleotides >90 bases may have interfering secondary structures.

Can I assemble linear fragments without a vector?
Yes. F1-X™ can assemble multiple linear fragments into complete plasmids, including entire constructs from synthetic DNA. Note that the orientation of overlap design dictates the final construct sequence.

How large can assembled fragments be?
For individual fragments: up to 32 kb. For total construct size: up to 100 kb. Larger constructs are possible with multi-stage assembly.

How many fragments can I assemble?
Up to 12 fragments is recommended for single-stage assembly, and we have verified up to 14 fragments in a single reaction. It is critical that overlaps are 40 bp or more for multi-fragment assemblies. More fragments may be possible with multi-stage approaches.

Will this work with repetitive sequences?
Yes. Design fragments with repetitive sequences internalized rather than at overlap regions, or use longer overlaps for uniqueness.

Can small fragments (≤200 bp) be assembled?
Yes. F1-X™ handles fragments as small as 100 bp. Use ≥5-fold molar excess for optimal results.

Do I need to use PCR for vector preparation?
No. Vectors can be linearized by restriction digest. All end types (blunt, 5′ overhang, 3′ overhang) are compatible. Vectors can also be prepared synthetically as gene blocks or fragments.

What are the shortest/longest overlaps I can use?
We recommend 40 bp as a starting point for all assemblies. For simple assemblies of 2–3 fragments, as little as 20 bp overlaps can work. For complex constructs of 4+ fragments, use 40+ bp.

How should I store the kit?
Store at -20°C in a stable temperature location (not a frost-free freezer). Limit to 5 freeze-thaw cycles or aliquot for frequent use.

What DNA concentrations should I use?
Use the amounts specified in the protocol section. Lower concentrations can be tested if materials are limiting.

Can I use different incubation times?
Use a minimum 15 minutes for simple assemblies; for more complex assemblies, we recommend starting with a 1 hour assembly time, then optimizing for shorter reaction times down to 15 minutes as allowed. We have seen success with multi-fragment assemblies (including the 4-fragment positive control and a 9-fragment control) down to 15-minute reaction times, but 60 minutes is a conservative place to start.

Can I use different incubation temperatures?
We recommend 50°C as a starting point for all assembly reactions. F1-X™ contains a proprietary blend of high-fidelity enzymes that are active between 50°C–56°C. If your construct requires additional optimization for enhanced annealing stringency, run the assembly at 53°C–56°C and extend the reaction time between 1–2 hours.

Do I need to inactivate restriction enzymes used to prep my DNA?
Yes. Residual restriction enzyme can cause star activity and DNA degradation.

Is gel purification necessary?
It is not required if PCR yields >80% full-length product. Low molecular weight contaminants caused by off-target amplification in PCR have a detrimental effect on cloning efficiency.

What sequencing protocol is recommended?
Sequence entire insert plus ~500 bp of vector at junctions. If PCR was used for vector preparation, consider sequencing entire construct using NGS or Sanger sequencing. We recommend consulting with your sequencing vendor to find the right workflow for your needs.

I don't see full-length product when I visualize assemblies via agarose gel electrophoresis. Is something wrong?
No, this is normal. Agarose gel electrophoresis is a qualitative, construct-dependent indicator of assembly success. Gibson Assembly reactions often produce high molecular weight species that appear as laddering rather than discrete bands. Compare your assembly reaction to a no-assembly control and look for reduced substrate bands and higher molecular weight products. For definitive confirmation, use transformation as your primary success metric — colony count and sequencing verification provide definitive confirmation of assembly success.