{"__v":0,"_id":"581a5b6a9d2fee0f005a2fa4","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"},"project":"5476bf0f817e8d080031f988","user":"568ed50cbeb2700d00471802","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":"2016-11-02T21:32:26.914Z","link_external":false,"link_url":"","githubsync":"","sync_unique":"","hidden":true,"api":{"settings":"","results":{"codes":[]},"auth":"required","params":[],"url":""},"isReference":false,"order":999,"body":"[block:api-header]\n{\n  \"type\": \"basic\",\n  \"title\": \"Description\"\n}\n[/block]\nCreate site-directed mutant plasmids quickly, efficiently and accurately. The protocol uses high fidelity enzyme technology to deliver fast and accurate mutagenesis of plasmids of up to 14 kb. Exclusive to the kit is a proprietary Pfu-based polymerase blend and an optimized Dpn I enzyme, which together allow for mutagenesis in approximately one hour, plus an overnight transformation. The protocol allows for synthesis of mutagenic oligonucleotides or the use of pre-existing mutagenic oligonucleotides.\n[block:api-header]\n{\n  \"type\": \"basic\",\n  \"title\": \"Sample requirements\"\n}\n[/block]\n## Plasmid to be mutagenized\n\nPlasmid DNA template must be isolated from a dam+ E. coli strain. Plasmid DNA isolated from dam– strains (e.g. JM110 and SCS110) is not suitable. It must be between 4000 and 14000bp in length and between 10 and 200ng/µL in concentration.\n\n## Mutagenic primers\n\nAll primers submitted in a single run must possess the same annealing temperature to the template.\n\n\n\n[block:api-header]\n{\n  \"type\": \"basic\",\n  \"title\": \"Required parameters\"\n}\n[/block]\n## Primer Source \n\nThere are 3 ways to provide mutagenic primers to a QuikChange lightning experiment largely depending on the number of mutants you are attempting to generate and whether you have the mutagenic primers already in stock.\n\n## Select from your inventory\n\nPrimer aliquots already in your inventory may be chosen for mutant generation.\n[block:callout]\n{\n  \"type\": \"info\",\n  \"title\": \"Mutagenic primers from your inventory\",\n  \"body\": \"Note when using mutagenic primers from your inventory the aliquot must have both Sequence and Concentration properties\"\n}\n[/block]\n\n[block:image]\n{\n  \"images\": [\n    {\n      \"image\": [\n        \"https://files.readme.io/7dd5f32-Transcriptic_2016-11-02_15-18-54.png\",\n        \"Transcriptic 2016-11-02 15-18-54.png\",\n        1152,\n        480,\n        \"#f5f5f6\"\n      ],\n      \"caption\": \"Example aliquot with appropriate properties for a mutagenic primer\"\n    }\n  ]\n}\n[/block]\n\n[block:parameters]\n{\n  \"data\": {\n    \"h-0\": \"Parameter\",\n    \"h-1\": \"Explanation\",\n    \"0-0\": \"Mutant Name\",\n    \"0-1\": \"A string of alphanumeric characters to identify the mutant.\",\n    \"1-0\": \"Primer 1\",\n    \"2-0\": \"Primer 2\",\n    \"1-1\": \"Aliquot, The forward mutagenic primer.\",\n    \"2-1\": \"Aliquot, The reverse mutagenic primer.\"\n  },\n  \"cols\": 2,\n  \"rows\": 3\n}\n[/block]\n## Individually input and synthesize new mutagenic oligos\n[block:parameters]\n{\n  \"data\": {\n    \"h-0\": \"Parameter\",\n    \"h-1\": \"Explanation\",\n    \"0-0\": \"Mutant Name\",\n    \"1-0\": \"Sequence\",\n    \"2-0\": \"Oligonucleotide Name\",\n    \"0-1\": \"Unique name for the mutant\",\n    \"1-1\": \"Primer sequence (GATCs allowed)\",\n    \"2-1\": \"Unique identifier for the oligonucleotide\"\n  },\n  \"cols\": 2,\n  \"rows\": 3\n}\n[/block]\n## Upload oligos for mutagenesis using a CSV file\n\nIn this mode, you simply upload a list of mutagenic oligonucleotides to be synthesised by the protocol. An example of the CSV format used is given below.\n[block:parameters]\n{\n  \"data\": {\n    \"h-0\": \"mutant_label\",\n    \"h-1\": \"oligo_label\",\n    \"h-2\": \"sequence\",\n    \"0-0\": \"mutant_1\",\n    \"0-1\": \"oligo_1\",\n    \"0-2\": \"atatatatatatatatatatatatatatatatatatatatatatatatatatatatat\",\n    \"1-0\": \"mutant_1\",\n    \"1-1\": \"oligo_2\",\n    \"1-2\": \"gggatatatatatatatatatatatatatatatatatatatatatatatatg\",\n    \"2-0\": \"mutant_2\",\n    \"2-1\": \"oligo_3\",\n    \"2-2\": \"ggctgatatatatatatatatatatatatatatatatatatatatatatatatatatat\",\n    \"3-0\": \"mutant_2\",\n    \"3-1\": \"oligo_4\",\n    \"3-2\": \"ggctgatatatatatatatatatatatatatatatatatatatatatatatatatatat\"\n  },\n  \"cols\": 3,\n  \"rows\": 4\n}\n[/block]\n## Plasmid to be mutagenized\n[block:callout]\n{\n  \"type\": \"warning\",\n  \"title\": \"Plasmid DNA template must be isolated from a dam+ E. coli strain. Plasmid DNA isolated from dam– strains (e.g. JM110 and SCS110) is not suitable\"\n}\n[/block]\n\n[block:parameters]\n{\n  \"data\": {\n    \"h-0\": \"Parameter\",\n    \"h-1\": \"Explanation\",\n    \"0-0\": \"Template DNA Plasmid\",\n    \"0-1\": \"Aliquot of DNA from your inventory\",\n    \"1-0\": \"Plasmid Length in BP\",\n    \"1-1\": \"The length of the plasmid in base pairs. This protocol has been optimized for plasmids in the range of 4000 to 14000bp\",\n    \"2-0\": \"Concentration (ng/µL)\",\n    \"2-1\": \"The concentration of the plasmid in ng/µL. Note this must be between 10 and 200 ng/µL\",\n    \"3-0\": \"Test 4 DNA template concentrations (100ng, 50ng, 25ng, 10ng)\",\n    \"3-1\": \"Checkbox, if you wish to try multiple template concentrations to optimize your mutagenesis, select this option. Otherwise, the protocol will just operate at a single concentration per protocol.\"\n  },\n  \"cols\": 2,\n  \"rows\": 4\n}\n[/block]\n\n[block:api-header]\n{\n  \"type\": \"basic\",\n  \"title\": \"Protocol Outputs\"\n}\n[/block]\nThere are multiple data and physical outputs from this protocol. Firstly a plate image and number of picked colonies is generated for every agar plate. \n\n## Containers\n[block:parameters]\n{\n  \"data\": {\n    \"h-0\": \"Container\",\n    \"h-1\": \"Container Type\",\n    \"h-2\": \"Storage\",\n    \"0-1\": \"96-flat\",\n    \"0-2\": \"cold_4\",\n    \"0-0\": \"96-well plate used for liquid cultures of picked colonies\",\n    \"1-0\": \"Mutagenic Primers\",\n    \"1-1\": \"micro-1.5 or 96-pcr (if number of primers is greater than 24)\",\n    \"1-2\": \"cold_20\"\n  },\n  \"cols\": 3,\n  \"rows\": 2\n}\n[/block]\n\n[block:callout]\n{\n  \"type\": \"warning\",\n  \"title\": \"Variable behaviour depending on mutagenic primer source\",\n  \"body\": \"Note that there is variable behaviour on whether containers will be created based on if the protocol is launched using primers already in inventory or primers being freshly synthesized.\"\n}\n[/block]\n## Datarefs\n[block:parameters]\n{\n  \"data\": {\n    \"h-0\": \"Dataref\",\n    \"0-0\": \"Autopick\",\n    \"h-1\": \"Type\",\n    \"0-1\": \"Annotated plate image, and a integer referring to the total number of colonies picked per autopick instruction\",\n    \"1-0\": \"Absorbance\",\n    \"1-1\": \"Colonies are picked and grown in a 96 well pick plate with LB and appropriate antibiotic. Following incubation the OD600 is measured.\"\n  },\n  \"cols\": 3,\n  \"rows\": 2\n}\n[/block]\n\n[block:api-header]\n{\n  \"type\": \"basic\",\n  \"title\": \"Recommended guidelines\"\n}\n[/block]\nFollowing the execution of this protocol you can use the outputs to perform a miniprep or Sanger sequencing step.\n[block:api-header]\n{\n  \"type\": \"basic\",\n  \"title\": \"Further reading\"\n}\n[/block]\nhttps://learn.transcriptic.com/quikchange-lightning/\n\nhttp://www.genomics.agilent.com/en/product.jsp?cid=AG-PT-175&tabId=AG-PR-1162&_requestid=1447350\n\nhttp://www.agilent.com/cs/library/usermanuals/Public/210518.pdf\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":"Agilent QuikChange Lightning Single","slug":"quikchange-lightning","type":"basic","title":"QuikChange Lightning"}

QuikChange Lightning

Agilent QuikChange Lightning Single

[block:api-header] { "type": "basic", "title": "Description" } [/block] Create site-directed mutant plasmids quickly, efficiently and accurately. The protocol uses high fidelity enzyme technology to deliver fast and accurate mutagenesis of plasmids of up to 14 kb. Exclusive to the kit is a proprietary Pfu-based polymerase blend and an optimized Dpn I enzyme, which together allow for mutagenesis in approximately one hour, plus an overnight transformation. The protocol allows for synthesis of mutagenic oligonucleotides or the use of pre-existing mutagenic oligonucleotides. [block:api-header] { "type": "basic", "title": "Sample requirements" } [/block] ## Plasmid to be mutagenized Plasmid DNA template must be isolated from a dam+ E. coli strain. Plasmid DNA isolated from dam– strains (e.g. JM110 and SCS110) is not suitable. It must be between 4000 and 14000bp in length and between 10 and 200ng/µL in concentration. ## Mutagenic primers All primers submitted in a single run must possess the same annealing temperature to the template. [block:api-header] { "type": "basic", "title": "Required parameters" } [/block] ## Primer Source There are 3 ways to provide mutagenic primers to a QuikChange lightning experiment largely depending on the number of mutants you are attempting to generate and whether you have the mutagenic primers already in stock. ## Select from your inventory Primer aliquots already in your inventory may be chosen for mutant generation. [block:callout] { "type": "info", "title": "Mutagenic primers from your inventory", "body": "Note when using mutagenic primers from your inventory the aliquot must have both Sequence and Concentration properties" } [/block] [block:image] { "images": [ { "image": [ "https://files.readme.io/7dd5f32-Transcriptic_2016-11-02_15-18-54.png", "Transcriptic 2016-11-02 15-18-54.png", 1152, 480, "#f5f5f6" ], "caption": "Example aliquot with appropriate properties for a mutagenic primer" } ] } [/block] [block:parameters] { "data": { "h-0": "Parameter", "h-1": "Explanation", "0-0": "Mutant Name", "0-1": "A string of alphanumeric characters to identify the mutant.", "1-0": "Primer 1", "2-0": "Primer 2", "1-1": "Aliquot, The forward mutagenic primer.", "2-1": "Aliquot, The reverse mutagenic primer." }, "cols": 2, "rows": 3 } [/block] ## Individually input and synthesize new mutagenic oligos [block:parameters] { "data": { "h-0": "Parameter", "h-1": "Explanation", "0-0": "Mutant Name", "1-0": "Sequence", "2-0": "Oligonucleotide Name", "0-1": "Unique name for the mutant", "1-1": "Primer sequence (GATCs allowed)", "2-1": "Unique identifier for the oligonucleotide" }, "cols": 2, "rows": 3 } [/block] ## Upload oligos for mutagenesis using a CSV file In this mode, you simply upload a list of mutagenic oligonucleotides to be synthesised by the protocol. An example of the CSV format used is given below. [block:parameters] { "data": { "h-0": "mutant_label", "h-1": "oligo_label", "h-2": "sequence", "0-0": "mutant_1", "0-1": "oligo_1", "0-2": "atatatatatatatatatatatatatatatatatatatatatatatatatatatatat", "1-0": "mutant_1", "1-1": "oligo_2", "1-2": "gggatatatatatatatatatatatatatatatatatatatatatatatatg", "2-0": "mutant_2", "2-1": "oligo_3", "2-2": "ggctgatatatatatatatatatatatatatatatatatatatatatatatatatatat", "3-0": "mutant_2", "3-1": "oligo_4", "3-2": "ggctgatatatatatatatatatatatatatatatatatatatatatatatatatatat" }, "cols": 3, "rows": 4 } [/block] ## Plasmid to be mutagenized [block:callout] { "type": "warning", "title": "Plasmid DNA template must be isolated from a dam+ E. coli strain. Plasmid DNA isolated from dam– strains (e.g. JM110 and SCS110) is not suitable" } [/block] [block:parameters] { "data": { "h-0": "Parameter", "h-1": "Explanation", "0-0": "Template DNA Plasmid", "0-1": "Aliquot of DNA from your inventory", "1-0": "Plasmid Length in BP", "1-1": "The length of the plasmid in base pairs. This protocol has been optimized for plasmids in the range of 4000 to 14000bp", "2-0": "Concentration (ng/µL)", "2-1": "The concentration of the plasmid in ng/µL. Note this must be between 10 and 200 ng/µL", "3-0": "Test 4 DNA template concentrations (100ng, 50ng, 25ng, 10ng)", "3-1": "Checkbox, if you wish to try multiple template concentrations to optimize your mutagenesis, select this option. Otherwise, the protocol will just operate at a single concentration per protocol." }, "cols": 2, "rows": 4 } [/block] [block:api-header] { "type": "basic", "title": "Protocol Outputs" } [/block] There are multiple data and physical outputs from this protocol. Firstly a plate image and number of picked colonies is generated for every agar plate. ## Containers [block:parameters] { "data": { "h-0": "Container", "h-1": "Container Type", "h-2": "Storage", "0-1": "96-flat", "0-2": "cold_4", "0-0": "96-well plate used for liquid cultures of picked colonies", "1-0": "Mutagenic Primers", "1-1": "micro-1.5 or 96-pcr (if number of primers is greater than 24)", "1-2": "cold_20" }, "cols": 3, "rows": 2 } [/block] [block:callout] { "type": "warning", "title": "Variable behaviour depending on mutagenic primer source", "body": "Note that there is variable behaviour on whether containers will be created based on if the protocol is launched using primers already in inventory or primers being freshly synthesized." } [/block] ## Datarefs [block:parameters] { "data": { "h-0": "Dataref", "0-0": "Autopick", "h-1": "Type", "0-1": "Annotated plate image, and a integer referring to the total number of colonies picked per autopick instruction", "1-0": "Absorbance", "1-1": "Colonies are picked and grown in a 96 well pick plate with LB and appropriate antibiotic. Following incubation the OD600 is measured." }, "cols": 3, "rows": 2 } [/block] [block:api-header] { "type": "basic", "title": "Recommended guidelines" } [/block] Following the execution of this protocol you can use the outputs to perform a miniprep or Sanger sequencing step. [block:api-header] { "type": "basic", "title": "Further reading" } [/block] https://learn.transcriptic.com/quikchange-lightning/ http://www.genomics.agilent.com/en/product.jsp?cid=AG-PT-175&tabId=AG-PR-1162&_requestid=1447350 http://www.agilent.com/cs/library/usermanuals/Public/210518.pdf [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]