{"__v":8,"_id":"572b8f654e904722003427c3","category":{"__v":17,"_id":"568f0e73bdb9260d00149d8c","pages":["568f0ea29ebef90d0087276a","568f0eafb700ce0d002f4a32","568f0ebbbdb9260d00149d8d","568f0edeb700ce0d002f4a34","568f0ef3bdb9260d00149d8f","568f12099ebef90d0087276d","568f121abeb2700d00471839","568f20e49ebef90d0087277e","568f446d9ebef90d0087278f","56c79a728bf67e0d00734767","56cb8830c675f50b00a4b834","56d47e99a4a9211b00c8f0d2","56d4af6c1c4de4130005d74e","56d52a131c4de4130005d80b","56de001b3db43f2000d9a765","56de1087f25ce60e002e31ad","56e314c46e602e0e00700b35"],"project":"5476bf0f817e8d080031f988","version":"5476bf10817e8d080031f98b","sync":{"url":"","isSync":false},"reference":false,"createdAt":"2016-01-08T01:18:43.343Z","from_sync":false,"order":1,"slug":"protocols","title":"Premade Protocols"},"parentDoc":null,"project":"5476bf0f817e8d080031f988","user":"56f57b8eab3f610e000a6596","version":{"__v":17,"_id":"5476bf10817e8d080031f98b","project":"5476bf0f817e8d080031f988","createdAt":"2014-11-27T06:05:04.263Z","releaseDate":"2014-11-27T06:05:04.263Z","categories":["5476bf10817e8d080031f98c","5477c46cf3736008009e9eb5","5477c474f3736008009e9eb6","5477c47ef3736008009e9eb7","5477c48ff3736008009e9eb8","5477c4948deb230800808bf0","54e68328154f8e0d0007b55c","54e90194c8e0c00d007ac061","54eed2275bf74a0d00ef4076","54f7a7be0a3cbb0d00d666fb","559b0ebf7ae7f80d0096d871","55d697f9ae529e0d00d34f03","562d4dcc8c6e5a0d00d6ed1d","562e591c4376430d006f17e0","568f0e73bdb9260d00149d8c","5719542aac1e2e0e001834c6","57a14a8ed778850e0047e230"],"is_deprecated":false,"is_hidden":false,"is_beta":false,"is_stable":true,"codename":"","version_clean":"1.0.0","version":"1.0"},"updates":[],"next":{"pages":[],"description":""},"createdAt":"2016-05-05T18:22:29.403Z","link_external":false,"link_url":"","githubsync":"","sync_unique":"","hidden":false,"api":{"results":{"codes":[]},"settings":"","auth":"required","params":[],"url":""},"isReference":false,"order":4,"body":"[block:api-header]\n{\n  \"type\": \"basic\",\n  \"title\": \"Description\"\n}\n[/block]\nThe Gibson Assembly protocol uses the Gibson Mix from [SGI-DNA](https://sgidna.com/reagents.html) to generate DNA plasmids from fragments with complimentary overlaps. Simple assemblies of small constructs with single inserts or complex assemblies of large constructs with multiple inserts can be accomplished with this protocol.\n\nThe protocol can be used for applications including multiple insert cloning, large fragment assemblies, and mutagenesis. Resulting constructs can be used in our [BasicPCR](doc:pcr), Transform, Spread, Pick, or Sequencing protocols.\n[block:api-header]\n{\n  \"type\": \"basic\",\n  \"title\": \"Sample requirements\"\n}\n[/block]\n\n[block:parameters]\n{\n  \"data\": {\n    \"h-0\": \"Sample\",\n    \"h-1\": \"Explanation\",\n    \"0-0\": \"Fragment\",\n    \"0-1\": \"A fragment with appropriate complimentary overlaps that you wish to assemble with other additional fragments into a single DNA plasmid. Fragment volumes must be greater than 1 uL.\"\n  },\n  \"cols\": 2,\n  \"rows\": 1\n}\n[/block]\n\n[block:api-header]\n{\n  \"type\": \"basic\",\n  \"title\": \"Required parameters\"\n}\n[/block]\n\n[block:parameters]\n{\n  \"data\": {\n    \"h-0\": \"Parameter\",\n    \"h-1\": \"Explanation\",\n    \"0-0\": \"Total Reaction Volume\",\n    \"0-1\": \"The total volume in which you would like your reaction to take place. Choose between 20 uL or 10 uL.\",\n    \"1-0\": \"Construct(s)\",\n    \"2-0\": \"Construct Name\",\n    \"1-1\": \"Each construct must have a unique name and use fragments from your Inventory. To add additional constructs, select 'Add Construct(s)' at the bottom of the current construct.\",\n    \"2-1\": \"A unique name for a single construct.\",\n    \"3-0\": \"Components\",\n    \"3-1\": \"The fragments and corresponding volumes that you wish to assemble into one construct. To add additional components, select 'Add Components' at the bottom of the current component. The total volume of components must not exceed 50% of the reaction volume.\",\n    \"4-0\": \"Assembly Conditions\",\n    \"4-1\": \"Specify the thermocycling conditions for your experiment. Please note that all constructs will undergo the same thermocycling conditions. If your constructs require separate conditions, please submit multiple runs.\"\n  },\n  \"cols\": 2,\n  \"rows\": 5\n}\n[/block]\n\n[block:api-header]\n{\n  \"type\": \"basic\",\n  \"title\": \"Protocol Outputs\"\n}\n[/block]\nThis protocol outputs a new aliquot of the assembled constructs which are stored in your inventory at -20°C. Any aliquots used in the making of the constructs will be returned to the storage condition from which they came.\n\nNo analytical or measurement data is returned by this protocol.\n[block:api-header]\n{\n  \"type\": \"basic\",\n  \"title\": \"Recommended guidelines\"\n}\n[/block]\n[SGI-DNA](https://sgidna.com/reagents.html) recommends this protocol for fragments ranging from 500 bp - 32 kb. The reaction should consist of 10-100ng of each DNA fragment, including the cloning vector, in equimolar amounts. Additional details on designing fragments, primers, and overlapping regions can be found on the SGI-DNA site [here](https://sgidna.com/hifi_kit.html) under \"Documentation\", \"User Manuals\" (see direct links to the manuals under \"Further reading\" below). \n\nEnsure you are aware of the dead volume in the containers you are using for the fragments. Dead volumes can be found in the containers section [here](https://developers.transcriptic.com/docs/containers#compatible-container-types). Failure to take into account dead volumes can result in insufficient volume being pipetted.\n[block:api-header]\n{\n  \"type\": \"basic\",\n  \"title\": \"Further reading\"\n}\n[/block]\n[SGI-DNA Gibson Assembly Guide](https://sgidna.com/dnamyway.html#GACGDownload)\n[Gibson Assembly HiFi 1 Step Kit](https://sgidna.com/hifi_kit.html)\n[Gibson Assembly HiFi 1-Step Kit Quick Reference Manual](https://sgidna.com/documentation/HiFi/Manual-HiFi/GA_HiFi_Quick-Ref.pdf)\n[Gibson Assembly HiFi 1-Step Kit Instructions](https://sgidna.com/documentation/HiFi/Manual-HiFi/GA_HiFi_Detailed_Manual.pdf)\n[block:callout]\n{\n  \"type\": \"success\",\n  \"body\": \"For further advice on your protocol please ask questions to the community at https://forum.transcriptic.com\",\n  \"title\": \"Check out the forum if you have more questions.\"\n}\n[/block]","excerpt":"","slug":"gibson-assembly","type":"basic","title":"Gibson Assembly"}
[block:api-header] { "type": "basic", "title": "Description" } [/block] The Gibson Assembly protocol uses the Gibson Mix from [SGI-DNA](https://sgidna.com/reagents.html) to generate DNA plasmids from fragments with complimentary overlaps. Simple assemblies of small constructs with single inserts or complex assemblies of large constructs with multiple inserts can be accomplished with this protocol. The protocol can be used for applications including multiple insert cloning, large fragment assemblies, and mutagenesis. Resulting constructs can be used in our [BasicPCR](doc:pcr), Transform, Spread, Pick, or Sequencing protocols. [block:api-header] { "type": "basic", "title": "Sample requirements" } [/block] [block:parameters] { "data": { "h-0": "Sample", "h-1": "Explanation", "0-0": "Fragment", "0-1": "A fragment with appropriate complimentary overlaps that you wish to assemble with other additional fragments into a single DNA plasmid. Fragment volumes must be greater than 1 uL." }, "cols": 2, "rows": 1 } [/block] [block:api-header] { "type": "basic", "title": "Required parameters" } [/block] [block:parameters] { "data": { "h-0": "Parameter", "h-1": "Explanation", "0-0": "Total Reaction Volume", "0-1": "The total volume in which you would like your reaction to take place. Choose between 20 uL or 10 uL.", "1-0": "Construct(s)", "2-0": "Construct Name", "1-1": "Each construct must have a unique name and use fragments from your Inventory. To add additional constructs, select 'Add Construct(s)' at the bottom of the current construct.", "2-1": "A unique name for a single construct.", "3-0": "Components", "3-1": "The fragments and corresponding volumes that you wish to assemble into one construct. To add additional components, select 'Add Components' at the bottom of the current component. The total volume of components must not exceed 50% of the reaction volume.", "4-0": "Assembly Conditions", "4-1": "Specify the thermocycling conditions for your experiment. Please note that all constructs will undergo the same thermocycling conditions. If your constructs require separate conditions, please submit multiple runs." }, "cols": 2, "rows": 5 } [/block] [block:api-header] { "type": "basic", "title": "Protocol Outputs" } [/block] This protocol outputs a new aliquot of the assembled constructs which are stored in your inventory at -20°C. Any aliquots used in the making of the constructs will be returned to the storage condition from which they came. No analytical or measurement data is returned by this protocol. [block:api-header] { "type": "basic", "title": "Recommended guidelines" } [/block] [SGI-DNA](https://sgidna.com/reagents.html) recommends this protocol for fragments ranging from 500 bp - 32 kb. The reaction should consist of 10-100ng of each DNA fragment, including the cloning vector, in equimolar amounts. Additional details on designing fragments, primers, and overlapping regions can be found on the SGI-DNA site [here](https://sgidna.com/hifi_kit.html) under "Documentation", "User Manuals" (see direct links to the manuals under "Further reading" below). Ensure you are aware of the dead volume in the containers you are using for the fragments. Dead volumes can be found in the containers section [here](https://developers.transcriptic.com/docs/containers#compatible-container-types). Failure to take into account dead volumes can result in insufficient volume being pipetted. [block:api-header] { "type": "basic", "title": "Further reading" } [/block] [SGI-DNA Gibson Assembly Guide](https://sgidna.com/dnamyway.html#GACGDownload) [Gibson Assembly HiFi 1 Step Kit](https://sgidna.com/hifi_kit.html) [Gibson Assembly HiFi 1-Step Kit Quick Reference Manual](https://sgidna.com/documentation/HiFi/Manual-HiFi/GA_HiFi_Quick-Ref.pdf) [Gibson Assembly HiFi 1-Step Kit Instructions](https://sgidna.com/documentation/HiFi/Manual-HiFi/GA_HiFi_Detailed_Manual.pdf) [block:callout] { "type": "success", "body": "For further advice on your protocol please ask questions to the community at https://forum.transcriptic.com", "title": "Check out the forum if you have more questions." } [/block]