{"_id":"572ba6b580b61a0e006afdb8","parentDoc":null,"project":"5476bf0f817e8d080031f988","user":"56f57b8eab3f610e000a6596","version":{"_id":"5476bf10817e8d080031f98b","__v":17,"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"},"category":{"_id":"568f0e73bdb9260d00149d8c","pages":["568f0ea29ebef90d0087276a","568f0eafb700ce0d002f4a32","568f0ebbbdb9260d00149d8d","568f0edeb700ce0d002f4a34","568f0ef3bdb9260d00149d8f","568f12099ebef90d0087276d","568f121abeb2700d00471839","568f20e49ebef90d0087277e","568f446d9ebef90d0087278f","56c79a728bf67e0d00734767","56cb8830c675f50b00a4b834","56d47e99a4a9211b00c8f0d2","56d4af6c1c4de4130005d74e","56d52a131c4de4130005d80b","56de001b3db43f2000d9a765","56de1087f25ce60e002e31ad","56e314c46e602e0e00700b35"],"__v":17,"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"},"__v":4,"updates":[],"next":{"pages":[],"description":""},"createdAt":"2016-05-05T20:01:57.125Z","link_external":false,"link_url":"","githubsync":"","sync_unique":"","hidden":true,"api":{"results":{"codes":[]},"settings":"","auth":"required","params":[],"url":""},"isReference":false,"order":7,"body":"[block:api-header]\n{\n  \"type\": \"basic\",\n  \"title\": \"Description\"\n}\n[/block]\nThe NEBuilder DNA Assembly protocol uses the NEBuilder Mix to assemble plasmids from fragments with complimentary overlaps. The mix consists of an exonuclease to create single-stranded 3' overhangs to facilitate the annealing of complementary fragments, polymerase to fill in the gaps within each annealed fragment, and a DNA ligase to seal nicks in the assembled DNA.\n\nThe resulting constructs can be used as templates for [BasicPCR](doc:pcr), RCA sequencing, or Transform, Spread, Pick 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]\nNew England Biolabs recommends that the concentration of fragments should be at least 2 times higher than the concentration of vector when assembling fragments into a cloning vector. For the fragments, PCR product purification is not necessary if the total volume of all PCR products is 20% or less of the assembly reaction volume. Overlaps on the fragments are recommended to be 15-80 bp. For more information on optimal fragment quantities, see \"NEBuilder HiFi DNA Assembly Reaction Protocol\" 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[NEBuilder HiFi DNA Assembly Master Mix](https://www.neb.com/products/e2621-nebuilder-hifi-dna-assembly-master-mix)\n[NEBuilder HiFi DNA Assembly Reaction Protocol](https://www.neb.com/protocols/2014/11/26/nebuilder-hifi-dna-assembly-reaction-protocol)\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":"nebuilder","type":"basic","title":"NEBuilder DNA Assembly"}

NEBuilder DNA Assembly


[block:api-header] { "type": "basic", "title": "Description" } [/block] The NEBuilder DNA Assembly protocol uses the NEBuilder Mix to assemble plasmids from fragments with complimentary overlaps. The mix consists of an exonuclease to create single-stranded 3' overhangs to facilitate the annealing of complementary fragments, polymerase to fill in the gaps within each annealed fragment, and a DNA ligase to seal nicks in the assembled DNA. The resulting constructs can be used as templates for [BasicPCR](doc:pcr), RCA sequencing, or Transform, Spread, Pick 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] New England Biolabs recommends that the concentration of fragments should be at least 2 times higher than the concentration of vector when assembling fragments into a cloning vector. For the fragments, PCR product purification is not necessary if the total volume of all PCR products is 20% or less of the assembly reaction volume. Overlaps on the fragments are recommended to be 15-80 bp. For more information on optimal fragment quantities, see "NEBuilder HiFi DNA Assembly Reaction Protocol" 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] [NEBuilder HiFi DNA Assembly Master Mix](https://www.neb.com/products/e2621-nebuilder-hifi-dna-assembly-master-mix) [NEBuilder HiFi DNA Assembly Reaction Protocol](https://www.neb.com/protocols/2014/11/26/nebuilder-hifi-dna-assembly-reaction-protocol) [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]