{"__v":5,"_id":"56de001b3db43f2000d9a765","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":"54eb9cdbdf7add210007b33e","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":"2016-03-07T22:26:35.225Z","link_external":false,"link_url":"","githubsync":"","sync_unique":"","hidden":false,"api":{"results":{"codes":[]},"settings":"","auth":"required","params":[],"url":""},"isReference":false,"order":19,"body":"[block:callout]\n{\n  \"type\": \"success\",\n  \"title\": \"This protocol is validated\",\n  \"body\": \"The validation document for this protocol is available here http://learn.transcriptic.com/rtqpcr/\"\n}\n[/block]\n\n[block:api-header]\n{\n  \"type\": \"basic\",\n  \"title\": \"Description\"\n}\n[/block]\nThis protocol allows you to assess the relative quantitation of genes in DNA samples. This protocol provides a master mix of polymerase, buffer and the florescent dye sybr green. All you need to provide is the DNA samples you would like to test and the forward and reverse primers. \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\": \"Template DNA\",\n    \"0-1\": \"For each reaction, select at least one aliquot of DNA. The optimal amount of cDNA to use in a single PCR is dependent upon the copy number of the target gene. We suggest using cDNA at a concentration of ~13ng/µl.\",\n    \"1-0\": \"Forward Primer\",\n    \"2-0\": \"Reverse Primer\",\n    \"1-1\": \"For each reaction, select a single forward primer. If you need to synthesize primers, please select the [oligosynthesis](https://developers.transcriptic.com/docs/oligosynthesis-1) protocol to do so. We suggest selecting primers with a concentration of 10µM. The forward and reverse primers concentration should be equimolar.\",\n    \"2-1\": \"For each reaction, also select a reverse primer. If you need to synthesize primers, please select the [oligosynthesis](https://developers.transcriptic.com/docs/oligosynthesis-1) protocol to do so. We suggest selecting primers with a concentration of 10µM.The forward and reverse primers concentration should be equimolar.\"\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\": \"Thermocycling conditions\",\n    \"0-1\": \"Specify the thermocycling conditions for your experiment. Please note that all reactions will undergo the same thermocycling conditions. If your primer sets require separate conditions, please submit multiple runs.\"\n  },\n  \"cols\": 2,\n  \"rows\": 1\n}\n[/block]\n\n[block:api-header]\n{\n  \"type\": \"basic\",\n  \"title\": \"Protocol Outputs\"\n}\n[/block]\nThis protocol returns a `.csv` file containing the results as list of each measurement obtained for each well at each time point. A visualization of this data is also returned on the results tab of the run. \n[block:api-header]\n{\n  \"type\": \"basic\",\n  \"title\": \"Recommended Guidelines\"\n}\n[/block]\nStandard curve: To assess absolute DNA concentrations, please include a sample with a known concentration to construct a standard curve. \n\nPCR controls: It is important to detect the presence of contaminating DNA that may affect the reliability of the data. Always include a no-template control (NTC) reaction, replacing the template with PCR grade water. \n[block:api-header]\n{\n  \"type\": \"basic\",\n  \"title\": \"Further reading\"\n}\n[/block]\nPlease see the  [oligosynthesis](https://developers.transcriptic.com/docs/oligosynthesis-1) protocol for primer synthesis. \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":"rtqpcr-1","type":"basic","title":"qPCR"}
[block:callout] { "type": "success", "title": "This protocol is validated", "body": "The validation document for this protocol is available here http://learn.transcriptic.com/rtqpcr/" } [/block] [block:api-header] { "type": "basic", "title": "Description" } [/block] This protocol allows you to assess the relative quantitation of genes in DNA samples. This protocol provides a master mix of polymerase, buffer and the florescent dye sybr green. All you need to provide is the DNA samples you would like to test and the forward and reverse primers. [block:api-header] { "type": "basic", "title": "Sample requirements" } [/block] [block:parameters] { "data": { "h-0": "Sample", "h-1": "Explanation", "0-0": "Template DNA", "0-1": "For each reaction, select at least one aliquot of DNA. The optimal amount of cDNA to use in a single PCR is dependent upon the copy number of the target gene. We suggest using cDNA at a concentration of ~13ng/µl.", "1-0": "Forward Primer", "2-0": "Reverse Primer", "1-1": "For each reaction, select a single forward primer. If you need to synthesize primers, please select the [oligosynthesis](https://developers.transcriptic.com/docs/oligosynthesis-1) protocol to do so. We suggest selecting primers with a concentration of 10µM. The forward and reverse primers concentration should be equimolar.", "2-1": "For each reaction, also select a reverse primer. If you need to synthesize primers, please select the [oligosynthesis](https://developers.transcriptic.com/docs/oligosynthesis-1) protocol to do so. We suggest selecting primers with a concentration of 10µM.The forward and reverse primers concentration should be equimolar." }, "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": "Thermocycling conditions", "0-1": "Specify the thermocycling conditions for your experiment. Please note that all reactions will undergo the same thermocycling conditions. If your primer sets require separate conditions, please submit multiple runs." }, "cols": 2, "rows": 1 } [/block] [block:api-header] { "type": "basic", "title": "Protocol Outputs" } [/block] This protocol returns a `.csv` file containing the results as list of each measurement obtained for each well at each time point. A visualization of this data is also returned on the results tab of the run. [block:api-header] { "type": "basic", "title": "Recommended Guidelines" } [/block] Standard curve: To assess absolute DNA concentrations, please include a sample with a known concentration to construct a standard curve. PCR controls: It is important to detect the presence of contaminating DNA that may affect the reliability of the data. Always include a no-template control (NTC) reaction, replacing the template with PCR grade water. [block:api-header] { "type": "basic", "title": "Further reading" } [/block] Please see the [oligosynthesis](https://developers.transcriptic.com/docs/oligosynthesis-1) protocol for primer synthesis. [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]