{"_id":"56c79a728bf67e0d00734767","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"},"parentDoc":null,"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"},"project":"5476bf0f817e8d080031f988","user":"568ed50cbeb2700d00471802","__v":18,"updates":[],"next":{"pages":[],"description":""},"createdAt":"2016-02-19T22:42:58.214Z","link_external":false,"link_url":"","githubsync":"","sync_unique":"","hidden":true,"api":{"results":{"codes":[]},"settings":"","auth":"required","params":[],"url":""},"isReference":false,"order":3,"body":"[block:callout]\n{\n  \"type\": \"success\",\n  \"title\": \"This is a validated protocol\",\n  \"body\": \"The validation document for this protocol is available here [http://learn.transcriptic.com/genotyping/](http://learn.transcriptic.com/genotyping/)\"\n}\n[/block]\n\n[block:api-header]\n{\n  \"type\": \"basic\",\n  \"title\": \"Description\"\n}\n[/block]\nGenotyping uses allele specific primers to identify the presence of an allele by performing PCR with template DNA taken from an organism. Most commonly this protocol is used for identifying the genotype of rats and mice via tail clippings.\n[block:api-header]\n{\n  \"type\": \"basic\",\n  \"title\": \"Sample requirements\"\n}\n[/block]\n\n[block:callout]\n{\n  \"type\": \"warning\",\n  \"title\": \"Sample aliquot lysis\",\n  \"body\": \"To specify a sample aliquot to be lysed prior to genotyping PCR, check the box next to the aliquot field. The lysis buffer is: (25mM NaOH, 0.2mM disodium EDTA) and neutralizing buffer (40mM Tris-HCl)\"\n}\n[/block]\n\n[block:parameters]\n{\n  \"data\": {\n    \"4-0\": \"Control Aliquots\",\n    \"h-0\": \"Sample\",\n    \"h-1\": \"Explanation\",\n    \"3-0\": \"Sample Aliquots\",\n    \"3-1\": \"A DNA or DNA containing tissue sample such as a mouse-tail clipping of unknown genotype. Lysis is optional.\",\n    \"4-1\": \"A DNA or DNA containing tissue sample such as a mouse-tail clipping of known genotype used as a positive or negative control. Lysis is optional\",\n    \"0-0\": \"Genotyping Reaction (Group)\",\n    \"0-1\": \"Select allele-specific primers and the corresponding DNA samples and controls for that primer pair.  For assessment of additional alleles, click 'Add Genotyping Reaction'.\\n\\nSamples must be submitted in Transcriptic provided 96-well PCR plates.\",\n    \"2-0\": \"Primer\",\n    \"2-1\": \"Choose an aliquot from your inventory that is a primer\",\n    \"1-0\": \"Primers (Group)\",\n    \"1-1\": \"Specify the primers to be used a single PCR genotyping.\"\n  },\n  \"cols\": 2,\n  \"rows\": 5\n}\n[/block]\n\n[block:api-header]\n{\n  \"type\": \"basic\",\n  \"title\": \"Required parameters\"\n}\n[/block]\n\n[block:callout]\n{\n  \"type\": \"warning\",\n  \"body\": \"For you to successfully execute a PCR genotyping run you must be sure of the parameters that you provide.\",\n  \"title\": \"Understand your parameters\"\n}\n[/block]\n\n[block:parameters]\n{\n  \"data\": {\n    \"h-0\": \"Parameter\",\n    \"h-1\": \"Explanation\",\n    \"0-0\": \"Primer Volume\",\n    \"0-1\": \"The volume of this primer included in each PCR reaction of 15 µL.  The recommended final concentration of each primer is 0.2-0.6 µM.\",\n    \"1-0\": \"Sample Volume / µL\",\n    \"1-1\": \"If providing liquid samples to be genotyped, specify the volume to be included in the PCR reaction. \\n\\n**Note: if providing solid samples for genotyping set this volume to 0.**\"\n  },\n  \"cols\": 2,\n  \"rows\": 2\n}\n[/block]\n## Polymerase type and thermocycling\n[block:parameters]\n{\n  \"data\": {\n    \"h-0\": \"Parameter\",\n    \"h-1\": \"Explanation\",\n    \"0-0\": \"Polymerase\",\n    \"0-1\": \"The DNA polymerase used in the PCR genotyping.\"\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]\n\n[block:parameters]\n{\n  \"data\": {\n    \"h-0\": \"Output\",\n    \"h-1\": \"Explanation\",\n    \"0-0\": \"Agarose gel electrophoretogram\",\n    \"0-1\": \"An annotated image resulting from the agarose gel electrophoresis of the genotyping PCR product is returned.\",\n    \"1-0\": \"Thermocycling parameters\",\n    \"1-1\": \"Much like you would be familiar with from a thermocycler. Groups of steps can be created for your full thermocycling process. Cycles for each group maybe specified on the left hand side.\"\n  },\n  \"cols\": 2,\n  \"rows\": 2\n}\n[/block]\n\n[block:api-header]\n{\n  \"type\": \"basic\",\n  \"title\": \"Recommended guidelines\"\n}\n[/block]\n\n[block:api-header]\n{\n  \"type\": \"basic\",\n  \"title\": \"Further reading\"\n}\n[/block]\n  * [Jackson Lab genotyping guide](https://www.jax.org/jax-mice-and-services/customer-support/technical-support/genotyping-resources/general-pcr-reminders-dos-and-donts) \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":"genotyping","type":"basic","title":"Genotyping"}
[block:callout] { "type": "success", "title": "This is a validated protocol", "body": "The validation document for this protocol is available here [http://learn.transcriptic.com/genotyping/](http://learn.transcriptic.com/genotyping/)" } [/block] [block:api-header] { "type": "basic", "title": "Description" } [/block] Genotyping uses allele specific primers to identify the presence of an allele by performing PCR with template DNA taken from an organism. Most commonly this protocol is used for identifying the genotype of rats and mice via tail clippings. [block:api-header] { "type": "basic", "title": "Sample requirements" } [/block] [block:callout] { "type": "warning", "title": "Sample aliquot lysis", "body": "To specify a sample aliquot to be lysed prior to genotyping PCR, check the box next to the aliquot field. The lysis buffer is: (25mM NaOH, 0.2mM disodium EDTA) and neutralizing buffer (40mM Tris-HCl)" } [/block] [block:parameters] { "data": { "4-0": "Control Aliquots", "h-0": "Sample", "h-1": "Explanation", "3-0": "Sample Aliquots", "3-1": "A DNA or DNA containing tissue sample such as a mouse-tail clipping of unknown genotype. Lysis is optional.", "4-1": "A DNA or DNA containing tissue sample such as a mouse-tail clipping of known genotype used as a positive or negative control. Lysis is optional", "0-0": "Genotyping Reaction (Group)", "0-1": "Select allele-specific primers and the corresponding DNA samples and controls for that primer pair. For assessment of additional alleles, click 'Add Genotyping Reaction'.\n\nSamples must be submitted in Transcriptic provided 96-well PCR plates.", "2-0": "Primer", "2-1": "Choose an aliquot from your inventory that is a primer", "1-0": "Primers (Group)", "1-1": "Specify the primers to be used a single PCR genotyping." }, "cols": 2, "rows": 5 } [/block] [block:api-header] { "type": "basic", "title": "Required parameters" } [/block] [block:callout] { "type": "warning", "body": "For you to successfully execute a PCR genotyping run you must be sure of the parameters that you provide.", "title": "Understand your parameters" } [/block] [block:parameters] { "data": { "h-0": "Parameter", "h-1": "Explanation", "0-0": "Primer Volume", "0-1": "The volume of this primer included in each PCR reaction of 15 µL. The recommended final concentration of each primer is 0.2-0.6 µM.", "1-0": "Sample Volume / µL", "1-1": "If providing liquid samples to be genotyped, specify the volume to be included in the PCR reaction. \n\n**Note: if providing solid samples for genotyping set this volume to 0.**" }, "cols": 2, "rows": 2 } [/block] ## Polymerase type and thermocycling [block:parameters] { "data": { "h-0": "Parameter", "h-1": "Explanation", "0-0": "Polymerase", "0-1": "The DNA polymerase used in the PCR genotyping." }, "cols": 2, "rows": 1 } [/block] [block:api-header] { "type": "basic", "title": "Protocol Outputs" } [/block] [block:parameters] { "data": { "h-0": "Output", "h-1": "Explanation", "0-0": "Agarose gel electrophoretogram", "0-1": "An annotated image resulting from the agarose gel electrophoresis of the genotyping PCR product is returned.", "1-0": "Thermocycling parameters", "1-1": "Much like you would be familiar with from a thermocycler. Groups of steps can be created for your full thermocycling process. Cycles for each group maybe specified on the left hand side." }, "cols": 2, "rows": 2 } [/block] [block:api-header] { "type": "basic", "title": "Recommended guidelines" } [/block] [block:api-header] { "type": "basic", "title": "Further reading" } [/block] * [Jackson Lab genotyping guide](https://www.jax.org/jax-mice-and-services/customer-support/technical-support/genotyping-resources/general-pcr-reminders-dos-and-donts) [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]