| type | | "Category:OSW81e9e22e7d934382a6a56df7d3736957" |
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| rdf_type | |
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| uuid | "2ae4e792-a1b5-4649-bedb-9ae90810567a" |
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| label | | text | "Homemade Gibson assembly mastermix" |
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| lang | "en" |
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| description | | text | "A 5X Gibson Assembly mastermix recipe and instructions for preparation, aliquoting and storage. The mix contains an exonuclease, a high-fidelity polymerase and a DNA ligase in a buffer that provides the required salts, cofactors and crowding agents. Enzyme activities (commercial units) should be adjusted according to vendor specifications and empirical performance. For best results prepare on ice, aliquot to avoid freeze-thaw cycles and store at -20°C." |
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| lang | "en" |
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| output_count | 1 |
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| preparation_duration | "30 min" |
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| processing_duration | "1 h" |
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| total_duration | "1 h 30 min" |
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| input | | "Tris-HCl pH 7.5 (buffer component; prepare from concentrated stock)" |
| "MgCl2 (magnesium source; prepare from concentrated stock)" |
| "Dithiothreitol (DTT) (reducing agent; prepare from 1 M stock)" |
| "NAD+ (cofactor for ligase; prepare from concentrated stock)" |
| "PEG-8000 (molecular crowding agent; prepare from concentrated stock)" |
| "dNTPs (equimolar mix of dATP, dTTP, dCTP, dGTP; use concentrated stock)" |
| "T5 exonuclease (or equivalent 5' exonuclease) — enzyme stock, add immediately before aliquoting or keep on ice during assembly" |
| "High-fidelity DNA polymerase (e.g. Phusion or equivalent) — enzyme stock" |
| "Thermostable DNA ligase (e.g. Taq DNA ligase) — enzyme stock" |
| "Nuclease-free water (to adjust volume)" |
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| steps | | @type | "schema:HowToStep" |
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| step_name | "Prepare buffer base" |
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| step_description | "On ice, combine the buffer components (Tris-HCl pH 7.5, MgCl2, DTT, NAD+, PEG-8000 and dNTPs) using concentrated stock solutions to produce a 5X master buffer. Mix gently by pipetting; avoid vortexing PEG-containing solutions." |
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| @type | "schema:HowToStep" |
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| step_name | "Add enzymes" |
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| step_description | "Keep enzymes on ice. Add the exonuclease, high-fidelity polymerase and thermostable ligase to the cold buffer so that the final combined preparation corresponds to a 5X mastermix (enzyme amounts adjusted from vendor unit activities). Mix by gentle pipetting." |
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| @type | "schema:HowToStep" |
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| step_name | "Aliquot and freeze" |
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| step_description | "Aliquot the completed 5X mastermix into small volumes (e.g., 20–50 µL) to avoid repeated freeze–thaw cycles. Flash-freeze on dry ice or in a -80°C freezer and store long-term at -20°C or -80°C depending on enzyme stability." |
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| @type | "schema:HowToStep" |
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| step_name | "Use in assembly reactions" |
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| step_description | "Thaw an aliquot on ice. For a 1X reaction, dilute the 5X mastermix to 1X with nuclease-free water and add DNA fragments at the desired molar ratios. Incubate according to the assembly protocol (typically 50°C for 15–60 min, depending on fragment complexity)." |
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| @type | "schema:HowToStep" |
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| step_name | "Quality control and troubleshooting" |
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| step_description | "Test the mastermix with a control assembly to confirm activity. If assembly yields are low, check enzyme activities, freshness of NAD+/dNTP stocks, correct pH of the buffer and incubation temperature/time." |
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| name | "HomemadeGibsonAssemblyMastermix" |
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