{"__v":5,"_id":"57228584de10810e0015398b","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":"2016-04-28T21:49:56.228Z","link_external":false,"link_url":"","githubsync":"","sync_unique":"","hidden":true,"api":{"results":{"codes":[]},"settings":"","auth":"required","params":[],"url":""},"isReference":false,"order":9,"body":"[block:api-header]\n{\n  \"type\": \"basic\",\n  \"title\": \"Description\"\n}\n[/block]\nThe Picogreen Assay protocol measures dsDNA concentration using PicoGreen dsDNA reagent. \n\nA Lambda DNA standard is included to create a standard curve with fluorescence measurements taken at an excitation wavelength of 485nm and an emission wavelength of 535nm. Before the fluorescence measurement is obtained, the PicoGreen is mixed with the samples and allowed to incubate at ambient temperature for 5 minutes while shaking. \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\": \"Samples\",\n    \"0-1\": \"Aliquots from your Inventory to be quantitated.\",\n    \"1-0\": \"\"\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\": \"Sample volume\",\n    \"1-0\": \"Standard curve\",\n    \"0-1\": \"Volume of sample aliquot to use in the assay.\",\n    \"1-1\": \"Select a standard curve from the options shown in the table below.\"\n  },\n  \"cols\": 2,\n  \"rows\": 2\n}\n[/block]\nChoose from the following options for a standard curve:\n\n[block:parameters]\n{\n  \"data\": {\n    \"h-0\": \"Standard\",\n    \"h-1\": \"DNA Amount Range\",\n    \"h-2\": \"Number of Wells\",\n    \"0-0\": \"All\",\n    \"0-1\": \"25pg - 1ug\",\n    \"0-2\": \"9\",\n    \"1-0\": \"Low\",\n    \"1-1\": \"25pg - 25ng\",\n    \"1-2\": \"5\",\n    \"2-0\": \"High\",\n    \"2-1\": \"25ng - 1ug\",\n    \"2-2\": \"5\"\n  },\n  \"cols\": 3,\n  \"rows\": 3\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 fluorescence measurements for the standard curve and each sample. Measurements are taken with an excitation wavelength of 485nm and an emission wavelength of 535nm. A visualization of measurement intensity is also provided in the run view under the 'Results' tab. \n[block:api-header]\n{\n  \"type\": \"basic\",\n  \"title\": \"Recommended guidelines\"\n}\n[/block]\n##Standard Curve\n\nThe standard curve should be selected to provide a DNA amount range which encompasses the expected DNA amount in the specified sample volume. Only three data points from the standards are required to create a standard curve, but five (including 0ng/uL) are provided to ensure that enough data points are available.\n\nUpon completion of the protocol, these data from the DNA standards should be plotted against the recorded fluorescence measurements. The equation for the line of best fit can then be used to calculate the amount of DNA present in the input samples.\n\n##Additional Guidelines\n\nIf the expected DNA concentration of a sample is higher than 1ug/mL, the sample should be diluted before measuring with this protocol.\n\nIf multiple sizes of DNA are present in a sample, gel purifying the target band prior to quantitation with this protocol is advised. If quantitating a PCR product, cleanup with the [ExoSAP-IT](doc:exosap-it) protocol prior to quantitation with this protocol is recommended.\n[block:api-header]\n{\n  \"type\": \"basic\",\n  \"title\": \"Further reading\"\n}\n[/block]\n[Quant-iT PicoGreen dsDNA Assay Kit](https://www.thermofisher.com/order/catalog/product/P7589) \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":"picogreen-assay","type":"basic","title":"Picogreen Assay"}
[block:api-header] { "type": "basic", "title": "Description" } [/block] The Picogreen Assay protocol measures dsDNA concentration using PicoGreen dsDNA reagent. A Lambda DNA standard is included to create a standard curve with fluorescence measurements taken at an excitation wavelength of 485nm and an emission wavelength of 535nm. Before the fluorescence measurement is obtained, the PicoGreen is mixed with the samples and allowed to incubate at ambient temperature for 5 minutes while shaking. [block:api-header] { "type": "basic", "title": "Sample requirements" } [/block] [block:parameters] { "data": { "h-0": "Sample", "h-1": "Explanation", "0-0": "Samples", "0-1": "Aliquots from your Inventory to be quantitated.", "1-0": "" }, "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": "Sample volume", "1-0": "Standard curve", "0-1": "Volume of sample aliquot to use in the assay.", "1-1": "Select a standard curve from the options shown in the table below." }, "cols": 2, "rows": 2 } [/block] Choose from the following options for a standard curve: [block:parameters] { "data": { "h-0": "Standard", "h-1": "DNA Amount Range", "h-2": "Number of Wells", "0-0": "All", "0-1": "25pg - 1ug", "0-2": "9", "1-0": "Low", "1-1": "25pg - 25ng", "1-2": "5", "2-0": "High", "2-1": "25ng - 1ug", "2-2": "5" }, "cols": 3, "rows": 3 } [/block] [block:api-header] { "type": "basic", "title": "Protocol Outputs" } [/block] This protocol returns a `.csv` file containing fluorescence measurements for the standard curve and each sample. Measurements are taken with an excitation wavelength of 485nm and an emission wavelength of 535nm. A visualization of measurement intensity is also provided in the run view under the 'Results' tab. [block:api-header] { "type": "basic", "title": "Recommended guidelines" } [/block] ##Standard Curve The standard curve should be selected to provide a DNA amount range which encompasses the expected DNA amount in the specified sample volume. Only three data points from the standards are required to create a standard curve, but five (including 0ng/uL) are provided to ensure that enough data points are available. Upon completion of the protocol, these data from the DNA standards should be plotted against the recorded fluorescence measurements. The equation for the line of best fit can then be used to calculate the amount of DNA present in the input samples. ##Additional Guidelines If the expected DNA concentration of a sample is higher than 1ug/mL, the sample should be diluted before measuring with this protocol. If multiple sizes of DNA are present in a sample, gel purifying the target band prior to quantitation with this protocol is advised. If quantitating a PCR product, cleanup with the [ExoSAP-IT](doc:exosap-it) protocol prior to quantitation with this protocol is recommended. [block:api-header] { "type": "basic", "title": "Further reading" } [/block] [Quant-iT PicoGreen dsDNA Assay Kit](https://www.thermofisher.com/order/catalog/product/P7589) [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]