F1-X™ Guidelines
| Parameter | Range |
|---|---|
| Fragment size | 100 bp – 32 kb per fragment |
| Fragment number | 2-12 fragments per reaction |
| Assembly size | Up to 100 kb total |
| Overlap length |
|
| Reaction conditions |
|
| Reaction temp. | 50°C (tolerance: 50-56°C) |
| Reaction volume | 20 μL (tolerance: 2.5-20 μL) |
| Compatibility |
|
| Storage temp. | -20 °C |
1. DNA Fragment Preparation and Quality Control
✓ Concentration: Quantify by fluorimetry or absorbance
✓ Integrity: Verify full-length products by electrophoresis. If <80% full-length product, consider gel extraction
✓ Purity: A260/280 ≥1.8, A260/230 ≥2.0. Crude PCR products may be used (≤20% v/v of reaction)
✓ Designed overlaps: Ensure homologous overlaps of ~40 bp are present on all DNA fragments
2. Calculate DNA Amounts and Prepare Fragment Mixes
Prepare DNA mixtures at >2× final target concentration.
| Simple Assemblies 2-3 Fragments | Complex Assemblies 4-12 Fragments |
|---|---|
| Total DNA*: 0.03-0.2 pmols | Total DNA*: 0.2-0.8 pmols |
| Vector: 50-100 ng per reaction | Per fragment: 0.02-0.1 pmol per reaction (0.05 target) |
| Ratio (Vector:Insert): 1:1-1:3, with 1:3 preferred* | Ratio: Equimolar for all |
*Final amounts in standard 20 μL reaction. For DNA fragments ≤100 bp, use 5× molar excess.
3. F1-X™ Assembly Procedure
Materials: F1-X™ Master Mix (2×), F1-X™ Positive Control (2×), Nuclease-free water, Thermocycler, DNA fragments
Assembly Protocol
1. Thaw F1-X™ Master Mix (2×) on ice
2. Vortex Master Mix vigorously for 15 seconds before use
3. Combine on ice: (see table below)
4. Mix thoroughly and spin down briefly
5. Incubate at 50°C for 15 mins for 3 frags or less, 60 mins for 4+ frags. If elevating incubation temp for complex assemblies (tolerance: 50-56°C), incubate for no less than 60 mins regardless of fragment number.
6. Store at -20°C or use immediately for transformation
| Component | Volume |
|---|---|
| F1-X™ Master Mix (2×) | 10 μL |
| DNA fragment mix* | X μL |
| Nuclease-free water | 10-X μL |
| Total reaction volume | 20 μL |
*if using F1-X™ Positive Control (2×), add 10 μL. No water needed.
Controls
• Positive Assembly, Vector Only, No-assembly
4. Quick Tips For Best Performance
User Guide
