{"_id":"573b5cb844685132006e3df2","parentDoc":null,"__v":5,"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"},"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"},"project":"5476bf0f817e8d080031f988","user":"56f57b8eab3f610e000a6596","updates":[],"next":{"pages":[],"description":""},"createdAt":"2016-05-17T18:02:32.226Z","link_external":false,"link_url":"","githubsync":"","sync_unique":"","hidden":true,"api":{"results":{"codes":[]},"settings":"","auth":"required","params":[],"url":""},"isReference":false,"order":21,"body":"[block:api-header]\n{\n  \"type\": \"basic\",\n  \"title\": \"Description\"\n}\n[/block]\nThe Sequencing protocol allows samples to be sequencing using dideoxysequencing or rolling circle amplification (RCA). For Dideoxy Sequencing, DNA samples are sequenced using Sanger chemistry. In RCA sequencing, rolling circle amplification is used to prepare samples, followed by Sanger sequencing. For both protocol modes, primers can be selected from your Inventory or the Transcriptic stock for convenience. The protocol returns sequencing data for your DNA or bacterial samples.\n\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\": \"DNA Samples\",\n    \"0-1\": \"Aliquots of DNA template to be sequenced using  dideoxysequencing.\",\n    \"1-0\": \"Bacterial Samples\",\n    \"2-0\": \"Primer (from Inventory)\",\n    \"1-1\": \"Aliquots of bacteria in bacterial broth to be sequenced after RCA preparation.\",\n    \"2-1\": \"Primers used in sequencing  DNA or bacterial samples.\"\n  },\n  \"cols\": 2,\n  \"rows\": 3\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\": \"Sequencing Reaction (group)\",\n    \"1-0\": \"RCA Bacteria/Primer pair (group)\",\n    \"2-0\": \"Sample Concentration (ng/uL)\",\n    \"4-0\": \"Stock Primer\",\n    \"3-0\": \"Primer Volume (from Inventory)\",\n    \"2-1\": \"Used to calculate the volume of DNA sample containing 500 ng of DNA to be used for Dideoxy Sequencing.\",\n    \"0-1\": \"A sequencing reaction group defines the primer for a set of DNA samples in Dideoxy Sequencing mode. To sequence samples with additional primers, create additional sequencing reactions by selecting 'Add Sequencing Reaction' at the bottom of the current group.\",\n    \"1-1\": \"A RCA bacteria/primer pair group defines the primer for a set of bacterial samples in RCA mode. To sequence samples with additional primers, create additional groups reactions by selecting 'Add RCA Bacteria/Primer pair' at the bottom of the current group.\",\n    \"4-1\": \"A primer from the Transcriptic stock to be used for sequencing (options are listed below).\",\n    \"3-1\": \"Volume of primer to be mixed with each DNA sample for Dideoxy Sequencing.\"\n  },\n  \"cols\": 2,\n  \"rows\": 5\n}\n[/block]\nCommonly used primers for sequencing MCS regions or other features can be provided by the Transcriptic reagent library. The current stock of primers includes the following list.\n[block:parameters]\n{\n  \"data\": {\n    \"h-0\": \"Primer\",\n    \"0-0\": \"T7 Forward\",\n    \"1-0\": \"T7 Reverse\",\n    \"2-0\": \"T3\",\n    \"3-0\": \"SP6\",\n    \"4-0\": \"M13 Reverse\",\n    \"5-0\": \"M13 Forward (-20)\",\n    \"6-0\": \"M13 Forward (-40)\",\n    \"7-0\": \"pGEX Forward\",\n    \"8-0\": \"pGEX Reverse\",\n    \"9-0\": \"BghRev\",\n    \"10-0\": \"ATTL-F\",\n    \"11-0\": \"ATTL-R\",\n    \"12-0\": \"pET\",\n    \"13-0\": \"T7 Forward (short)\",\n    \"14-0\": \"gSP6\",\n    \"15-0\": \"KBR/TJ\",\n    \"16-0\": \"EGFP-C\",\n    \"17-0\": \"EGFP-N\",\n    \"18-0\": \"CMV-F\"\n  },\n  \"cols\": 1,\n  \"rows\": 19\n}\n[/block]\n\n[block:api-header]\n{\n  \"type\": \"basic\",\n  \"title\": \"Protocol Outputs\"\n}\n[/block]\nData is returned from the Sanger sequencing of the DNA or bacterial samples. The sequencing data is returned as a zip file of `.ab1` and `.seq` files.\n[block:api-header]\n{\n  \"type\": \"basic\",\n  \"title\": \"Recommended guidelines\"\n}\n[/block]\nFor Dideoxy Sequencing, ensure that 500 ng of DNA is present in each DNA sample aliquot. 1uL of 20uM primer is recommended as the primer volume, to be used in a total reaction volume of 15uL.\n\nFor RCA sequencing, the bacterial samples must have a volume of at least 30uL. A minimum of 5uL of 20uM primer is required in a tube container. The primer tube will be sent with the bacterial samples for sequencing and returned to the storage condition from which it came when sequencing is complete.\n[block:api-header]\n{\n  \"type\": \"basic\",\n  \"title\": \"Further reading\"\n}\n[/block]\nFor more information, please see the [Sanger Sequencing](doc:sanger-sequencing) capabilities page and the [Elim Biopharm](http://www.elimbio.com/dna_sequencing.htm) site.\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":"sequencing","type":"basic","title":"Sequencing"}
[block:api-header] { "type": "basic", "title": "Description" } [/block] The Sequencing protocol allows samples to be sequencing using dideoxysequencing or rolling circle amplification (RCA). For Dideoxy Sequencing, DNA samples are sequenced using Sanger chemistry. In RCA sequencing, rolling circle amplification is used to prepare samples, followed by Sanger sequencing. For both protocol modes, primers can be selected from your Inventory or the Transcriptic stock for convenience. The protocol returns sequencing data for your DNA or bacterial samples. [block:api-header] { "type": "basic", "title": "Sample requirements" } [/block] [block:parameters] { "data": { "h-0": "Sample", "h-1": "Explanation", "0-0": "DNA Samples", "0-1": "Aliquots of DNA template to be sequenced using dideoxysequencing.", "1-0": "Bacterial Samples", "2-0": "Primer (from Inventory)", "1-1": "Aliquots of bacteria in bacterial broth to be sequenced after RCA preparation.", "2-1": "Primers used in sequencing DNA or bacterial samples." }, "cols": 2, "rows": 3 } [/block] [block:api-header] { "type": "basic", "title": "Required parameters" } [/block] [block:parameters] { "data": { "h-0": "Parameter", "h-1": "Explanation", "0-0": "Sequencing Reaction (group)", "1-0": "RCA Bacteria/Primer pair (group)", "2-0": "Sample Concentration (ng/uL)", "4-0": "Stock Primer", "3-0": "Primer Volume (from Inventory)", "2-1": "Used to calculate the volume of DNA sample containing 500 ng of DNA to be used for Dideoxy Sequencing.", "0-1": "A sequencing reaction group defines the primer for a set of DNA samples in Dideoxy Sequencing mode. To sequence samples with additional primers, create additional sequencing reactions by selecting 'Add Sequencing Reaction' at the bottom of the current group.", "1-1": "A RCA bacteria/primer pair group defines the primer for a set of bacterial samples in RCA mode. To sequence samples with additional primers, create additional groups reactions by selecting 'Add RCA Bacteria/Primer pair' at the bottom of the current group.", "4-1": "A primer from the Transcriptic stock to be used for sequencing (options are listed below).", "3-1": "Volume of primer to be mixed with each DNA sample for Dideoxy Sequencing." }, "cols": 2, "rows": 5 } [/block] Commonly used primers for sequencing MCS regions or other features can be provided by the Transcriptic reagent library. The current stock of primers includes the following list. [block:parameters] { "data": { "h-0": "Primer", "0-0": "T7 Forward", "1-0": "T7 Reverse", "2-0": "T3", "3-0": "SP6", "4-0": "M13 Reverse", "5-0": "M13 Forward (-20)", "6-0": "M13 Forward (-40)", "7-0": "pGEX Forward", "8-0": "pGEX Reverse", "9-0": "BghRev", "10-0": "ATTL-F", "11-0": "ATTL-R", "12-0": "pET", "13-0": "T7 Forward (short)", "14-0": "gSP6", "15-0": "KBR/TJ", "16-0": "EGFP-C", "17-0": "EGFP-N", "18-0": "CMV-F" }, "cols": 1, "rows": 19 } [/block] [block:api-header] { "type": "basic", "title": "Protocol Outputs" } [/block] Data is returned from the Sanger sequencing of the DNA or bacterial samples. The sequencing data is returned as a zip file of `.ab1` and `.seq` files. [block:api-header] { "type": "basic", "title": "Recommended guidelines" } [/block] For Dideoxy Sequencing, ensure that 500 ng of DNA is present in each DNA sample aliquot. 1uL of 20uM primer is recommended as the primer volume, to be used in a total reaction volume of 15uL. For RCA sequencing, the bacterial samples must have a volume of at least 30uL. A minimum of 5uL of 20uM primer is required in a tube container. The primer tube will be sent with the bacterial samples for sequencing and returned to the storage condition from which it came when sequencing is complete. [block:api-header] { "type": "basic", "title": "Further reading" } [/block] For more information, please see the [Sanger Sequencing](doc:sanger-sequencing) capabilities page and the [Elim Biopharm](http://www.elimbio.com/dna_sequencing.htm) site. [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]