{"_id":"552dfaca2594f70d001b2c4a","__v":17,"project":"5476bf0f817e8d080031f988","user":"5476beec817e8d080031f986","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":"54f7a7be0a3cbb0d00d666fb","version":"5476bf10817e8d080031f98b","__v":19,"project":"5476bf0f817e8d080031f988","pages":["54f7a95354182d2100c9d057","54f7ad293607243500de2496","54f7ae7d0a3cbb0d00d66705","54f7b0013607243500de249b","54f7b4360a3cbb0d00d6670c","54f7c71154182d2100c9d073","54fa5205961fea21009206a9","54fa55e1c6db4517005b0147","54fddf642804410d00ee8a2a","5509fd2bdfed731900b81863","552dfa862594f70d001b2c48","552dfab3a702770d00d96d5b","552dfaca2594f70d001b2c4a","55d68d2c250d7d0d00427478","55d68d3bae529e0d00d34edb","5611dd433ca69417008981af","5679bca976cd370d003c1183","56de30df9ca83e17000cbc59","56eadf62199f7317006eb4db"],"sync":{"url":"","isSync":false},"reference":false,"createdAt":"2015-03-05T00:47:58.582Z","from_sync":false,"order":4,"slug":"instructions","title":"Capabilities"},"parentDoc":null,"updates":[],"next":{"pages":[],"description":""},"createdAt":"2015-04-15T05:44:42.662Z","link_external":false,"link_url":"","githubsync":"","sync_unique":"","hidden":false,"api":{"results":{"codes":[]},"settings":"","auth":"required","params":[],"url":""},"isReference":false,"order":18,"body":"[block:api-header]\n{\n  \"type\": \"basic\",\n  \"title\": \"Instruction and Parameters\"\n}\n[/block]\n`sanger_sequence` specifies which well indices of a container (`object`) should be sent for sequencing. The wells specified should already contain the DNA to be sequenced and sequencing primer(s) in their respective appropriate concentrations. \n[block:code]\n{\n  \"codes\": [\n    {\n      \"code\": \"{\\n  \\\"op\\\": \\\"sanger_sequence\\\",\\n  \\\"type\\\": \\\"standard\\\" | \\\"RCA\\\",\\n  \\\"object\\\": container,\\n  \\\"wells\\\": [well_index],\\n  \\\"primer\\\": container, // only required for RCA\\n  \\\"dataref\\\": string\\n}\",\n      \"language\": \"json\",\n      \"name\": \"sanger_sequence\"\n    }\n  ]\n}\n[/block]\nTwo types of sequencing are available, `\"standard\"` and `\"RCA\"` (rolling circle amplification). Standard sequencing uses Sanger sequencing to sequence DNA templates with appropriate primers. In contrast, specifying `\"RCA\"` will result in the use of rolling circle amplification to prepare DNA samples, followed by Sanger sequencing. Samples for RCA sequencing should be submitted as aliquots of bacterial broth with a minimum volume of 30uL.\n[block:api-header]\n{\n  \"type\": \"basic\",\n  \"title\": \"Device\"\n}\n[/block]\nTranscriptic currently uses an outside service (Elim) for its sequencing needs.\n\nEach well specified for a `\"standard\"` sequencing reaction should have a total volume of **15 µl**. We recommend that each of these wells contains the following composition of DNA and primer, premixed together:\n[block:parameters]\n{\n  \"data\": {\n    \"h-0\": \"Template Type\",\n    \"h-1\": \"Amount of DNA\",\n    \"h-2\": \"Amount of Primer\",\n    \"h-3\": \"Total Volume (DNA+Primer)\",\n    \"0-3\": \"15µl\",\n    \"1-3\": \"15µl\",\n    \"2-3\": \"15µl\",\n    \"3-3\": \"15µl\",\n    \"0-2\": \"1 µl of a 20 µM stock\",\n    \"1-2\": \"1 µl of a 20 µM stock\",\n    \"2-2\": \"\",\n    \"3-2\": \"9 pmol\",\n    \"0-1\": \"500 ng\",\n    \"1-1\": \"40 ng\",\n    \"2-1\": \"30 ng\",\n    \"3-1\": \"50 ng\",\n    \"0-0\": \"Plasmid\",\n    \"1-0\": \"*PCR product\",\n    \"2-0\": \"PCR (500bp - 1kb)\",\n    \"3-0\": \"PCR (>1kb)\"\n  },\n  \"cols\": 3,\n  \"rows\": 2\n}\n[/block]\n*It is recommended that PCR products are cleaned up using a method like ExoSap Purification before sequencing.\n\nEach well specified for an `\"RCA\"` sequencing reaction should have a total volume of **30 µl**, and should contain liquid culture media (with antibiotic) that has been innoculated with a single bacterial clone. Each `\"RCA\"` sequencing instruction should also be accompanied by a separate tube containing your primer, as specified in the `\"primer\"` field. We recommend that each sample well contains the following composition of bacteria, and that each primer tube contains the following composition of primer (primer and bacteria should be kept separate):\n[block:parameters]\n{\n  \"data\": {\n    \"h-0\": \"Template Type\",\n    \"h-1\": \"Amount of Bacteria\",\n    \"h-2\": \"Amount of Primer\",\n    \"0-0\": \"Bacterial culture (for RCA sequencing)\",\n    \"0-1\": \"30 µl\",\n    \"0-2\": \"1 µl of a 20 µM stock\"\n  },\n  \"cols\": 3,\n  \"rows\": 1\n}\n[/block]\n*We recommend that each primer tube contains at least 5 µl of primer, even if it will be used for less than 5 sequencing reactions. We also recommend that each `\"primer\"` tube should be prepared as a fresh aliquot, immediately before the sequencing instruction, by making a dilution from your higher concentration primer stocks.\n\nIn the returned dataset, sequencing traces will be labelled with the `dataref` and well id.\n\nMore information can be found on the [ELIM Biopharm site](http://www.elimbio.com/dna_sequencing.htm).","excerpt":"","slug":"sanger-sequencing","type":"basic","title":"Sanger Sequencing"}
[block:api-header] { "type": "basic", "title": "Instruction and Parameters" } [/block] `sanger_sequence` specifies which well indices of a container (`object`) should be sent for sequencing. The wells specified should already contain the DNA to be sequenced and sequencing primer(s) in their respective appropriate concentrations. [block:code] { "codes": [ { "code": "{\n \"op\": \"sanger_sequence\",\n \"type\": \"standard\" | \"RCA\",\n \"object\": container,\n \"wells\": [well_index],\n \"primer\": container, // only required for RCA\n \"dataref\": string\n}", "language": "json", "name": "sanger_sequence" } ] } [/block] Two types of sequencing are available, `"standard"` and `"RCA"` (rolling circle amplification). Standard sequencing uses Sanger sequencing to sequence DNA templates with appropriate primers. In contrast, specifying `"RCA"` will result in the use of rolling circle amplification to prepare DNA samples, followed by Sanger sequencing. Samples for RCA sequencing should be submitted as aliquots of bacterial broth with a minimum volume of 30uL. [block:api-header] { "type": "basic", "title": "Device" } [/block] Transcriptic currently uses an outside service (Elim) for its sequencing needs. Each well specified for a `"standard"` sequencing reaction should have a total volume of **15 µl**. We recommend that each of these wells contains the following composition of DNA and primer, premixed together: [block:parameters] { "data": { "h-0": "Template Type", "h-1": "Amount of DNA", "h-2": "Amount of Primer", "h-3": "Total Volume (DNA+Primer)", "0-3": "15µl", "1-3": "15µl", "2-3": "15µl", "3-3": "15µl", "0-2": "1 µl of a 20 µM stock", "1-2": "1 µl of a 20 µM stock", "2-2": "", "3-2": "9 pmol", "0-1": "500 ng", "1-1": "40 ng", "2-1": "30 ng", "3-1": "50 ng", "0-0": "Plasmid", "1-0": "*PCR product", "2-0": "PCR (500bp - 1kb)", "3-0": "PCR (>1kb)" }, "cols": 3, "rows": 2 } [/block] *It is recommended that PCR products are cleaned up using a method like ExoSap Purification before sequencing. Each well specified for an `"RCA"` sequencing reaction should have a total volume of **30 µl**, and should contain liquid culture media (with antibiotic) that has been innoculated with a single bacterial clone. Each `"RCA"` sequencing instruction should also be accompanied by a separate tube containing your primer, as specified in the `"primer"` field. We recommend that each sample well contains the following composition of bacteria, and that each primer tube contains the following composition of primer (primer and bacteria should be kept separate): [block:parameters] { "data": { "h-0": "Template Type", "h-1": "Amount of Bacteria", "h-2": "Amount of Primer", "0-0": "Bacterial culture (for RCA sequencing)", "0-1": "30 µl", "0-2": "1 µl of a 20 µM stock" }, "cols": 3, "rows": 1 } [/block] *We recommend that each primer tube contains at least 5 µl of primer, even if it will be used for less than 5 sequencing reactions. We also recommend that each `"primer"` tube should be prepared as a fresh aliquot, immediately before the sequencing instruction, by making a dilution from your higher concentration primer stocks. In the returned dataset, sequencing traces will be labelled with the `dataref` and well id. More information can be found on the [ELIM Biopharm site](http://www.elimbio.com/dna_sequencing.htm).