{"__v":26,"_id":"568f446d9ebef90d0087278f","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":"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":"2016-01-08T05:09:01.411Z","link_external":false,"link_url":"","githubsync":"","sync_unique":"","hidden":false,"api":{"results":{"codes":[]},"settings":"","auth":"required","params":[],"url":""},"isReference":false,"order":5,"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/kunkel-mutagenesis/\"\n}\n[/block]\n\n[block:api-header]\n{\n  \"type\": \"basic\",\n  \"title\": \"Description\"\n}\n[/block]\nMutate and generate select DNA plasmids using Kunkel Mutagenesis. This protocol takes a target single stranded DNA molecule and anneals it to a complimentary nucleotide with a base mis-match. DNA polymerase then fills in the missing bases making a double stranded DNA molecule where the antisense strand carries the point mutation. From here DNA replication can take place replicating a sense strand that carries the desired point mutation from the oligonucleotide. 1 or more mutant constructs can be specified with one or more mutations to be made per construct.\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    \"0-1\": \"A single aliquot of a solution of single stranded DNA. Usually this ssDNA will contain the element (gene, promoter, etc.) you wish to impart a mutation to.\",\n    \"0-0\": \"ssDNA\",\n    \"1-0\": \"RCA sequencing primers (optional)\",\n    \"2-0\": \"Mutant Oligonucleotides (optional)\",\n    \"1-1\": \"If sequencing mutant constructs with non-library standard primers they will be required for this protocol.\",\n    \"2-1\": \"If you wish to reuse a oligonucleotide to impart a mutation from a previous Kunkel Mutagenesis run, they can be used in future runs.\"\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##Construct Parameters\n[block:parameters]\n{\n  \"data\": {\n    \"h-0\": \"Parameter\",\n    \"h-1\": \"Explanation\",\n    \"0-0\": \"Number of colonies to pick\",\n    \"1-0\": \"Antibiotic resistance\",\n    \"0-1\": \"An integer that specifies how many colonies you would like to be picked per mutant generated. Each picked colony will undergo growth and sequencing\",\n    \"1-1\": \"Antibiotic to supplement growth media and agar with for colony growth. Choose from Kanamycin, Ampicillin and Spectinomycin\"\n  },\n  \"cols\": 2,\n  \"rows\": 2\n}\n[/block]\n##Rolling Circle Amplification (RCA) Sequencing Primer(s)\n[block:callout]\n{\n  \"type\": \"info\",\n  \"title\": \"Choose you primer source\",\n  \"body\": \"For sequencing you can choose from stock primers provided by Transcriptic or primers in your inventory.\"\n}\n[/block]\n\n[block:parameters]\n{\n  \"data\": {\n    \"h-0\": \"Parameter\",\n    \"h-1\": \"Explanation\",\n    \"0-0\": \"Sequencing Primers\",\n    \"0-1\": \"Primers used for sequencing your mutant constructs to confirm the mutation. More than one primer may be specified to sequence multiple regions of the construct. Average read length for the sequencing is approximately 800-900bp.\"\n  },\n  \"cols\": 2,\n  \"rows\": 1\n}\n[/block]\n### Stock Primers\nCommonly used primers for sequencing MCS regions or other features can be provided by the Transcriptic reagent library. The current stock of primers includes the following list.\n[block:parameters]\n{\n  \"data\": {\n    \"h-0\": \"Primer Name\",\n    \"0-0\": \"T7 Forward\",\n    \"1-0\": \"T7 Reverse\",\n    \"2-0\": \"T3\",\n    \"3-0\": \"M13 Reverse\",\n    \"4-0\": \"M13 Forward (-20)\",\n    \"5-0\": \"M13 Forward (-40)\",\n    \"6-0\": \"pGEX Forward\",\n    \"7-0\": \"pGEX Reverse\",\n    \"8-0\": \"BghRev\",\n    \"9-0\": \"ATTL-F\",\n    \"10-0\": \"ATTL-R\",\n    \"11-0\": \"pET\",\n    \"12-0\": \"T7 Forward (short)\",\n    \"13-0\": \"gSP6\",\n    \"14-0\": \"KBR/TJ\",\n    \"15-0\": \"EGFP-C\",\n    \"16-0\": \"EGFP-N\",\n    \"17-0\": \"CMV-F\"\n  },\n  \"cols\": 1,\n  \"rows\": 18\n}\n[/block]\n##Mutant Constructs\n\nOne or more mutant construct can be specified with one or more mutations. To add another mutant construct click the 'Add Mutant' link.\n[block:callout]\n{\n  \"type\": \"info\",\n  \"title\": \"Specify the mutant constructs desired\",\n  \"body\": \"Mutant constructs can specified in 3 ways:\\n\\n1. Bulk upload of sequences by a `.csv` file\\n2. Sequence specified for synthesis during the run\\n3. Aliquots from your inventory can be provided. \\n\\nA CSV template is available for CSV upload from the Protocol Browser.\"\n}\n[/block]\n### `.csv` upload\nUse this functionality if you are specifying many mutant constructs at once.\n[block:parameters]\n{\n  \"data\": {\n    \"h-0\": \"Parameter\",\n    \"h-1\": \"Explanation\",\n    \"0-0\": \"Mutant constructs\",\n    \"0-1\": \"Click 'Upload CSV' to upload a `.csv` file describing your mutant constructs. A template for the CSV file can be downloaded from the Protocol Browser.\"\n  },\n  \"cols\": 2,\n  \"rows\": 1\n}\n[/block]\n### Enter mutant parameters\nUse this functionality if you are specifying a small number of mutants.\n[block:parameters]\n{\n  \"data\": {\n    \"0-0\": \"Mutant (Group)\",\n    \"0-1\": \"Multiple mutants maybe specified, additional mutants can be added by clicking the 'Add Mutant' link at the bottom of the mutant group.\\n\\nA mutant group contains the fields `Mutant Name` and `Oligonucleotides`.\",\n    \"1-0\": \"Mutant Name\",\n    \"1-1\": \"The name that you wish to assign to the resulting mutant construct.\",\n    \"2-0\": \"Oligonucleotides (Group)\",\n    \"3-0\": \"Sequence\",\n    \"4-0\": \"Oligo Label\",\n    \"2-1\": \"1 or more oligo nucleotides used to insert mutations to a target sequence through a base pair mismatch. To add more oligonucleotides click the 'Add Oligonucleotides' link at the bottom of the Oligonucleotides group.\",\n    \"3-1\": \"The sequence of the oligonucleotide that will be used to introduce a mutation.\",\n    \"4-1\": \"The label you wish to assign to that oligonucleotide.\",\n    \"h-0\": \"Parameter\",\n    \"h-1\": \"Explanation\"\n  },\n  \"cols\": 2,\n  \"rows\": 5\n}\n[/block]\n### Enter Mutant Parameters Selecting Oligos From Your Inventory\n[block:parameters]\n{\n  \"data\": {\n    \"h-0\": \"Parameter\",\n    \"h-1\": \"Explanation\",\n    \"0-0\": \"Mutant Name\",\n    \"0-1\": \"The name you would like to assign to this mutant.\",\n    \"1-0\": \"Oligonucleotides\",\n    \"1-1\": \"1 or more oligonucleotide aliquots used to insert mutations to a target sequence through a base pair mismatch.\"\n  },\n  \"cols\": 2,\n  \"rows\": 2\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\": \"Plate image\",\n    \"1-0\": \"Picked colonies\",\n    \"2-0\": \"Sequencing data (optional)\",\n    \"0-1\": \"A plate image is returned of all LB-agar cultures for each of the mutants.\",\n    \"1-1\": \"The number of colonies picked per well in the LB agar plates is returned.\",\n    \"2-1\": \"Data is returned from the Sanger sequencing of the mutant constructs. The sequencing data is returned as a zip file of `.ab1` and `.seq` files\"\n  },\n  \"cols\": 2,\n  \"rows\": 3\n}\n[/block]\n\n[block:api-header]\n{\n  \"type\": \"basic\",\n  \"title\": \"Recommended guidelines\"\n}\n[/block]\n ## ssDNA\n  * ssDNA concentration should be in the range of  30-40ng/µL (the protocol uses 2µL of ssDNA per construct); \n  * ssDNA should be sent to Transcriptic via a sample intake kit and shipped on dry ice and storage of -20C should be specified. no recommendations on length or GC content apart from standard PCR rules since the rxn uses a T7 polymerase. \n  * Typical successful  plasmids have been in the size range of ~6.5-7kb\n  * For calculating the volume of ssDNA required per run, the following equation should be used to take into account container dead volumes:\n\n       ```(number_of_reactions + 3) * 1.5 * 2µL```\n\n       For 2 reactions this would be:\n\n       ```(2 + 3) * 1.5 * 2µL = 15µL```\n\n## General\n  * Mutation oligonucleotides are recommend to approximately 33 bp for single mutations. Longer oligonucleotides are necessary if they contain more than 1 mutation site. The oligonucleotides should not overlap. If supplying oligonucleotides from your inventory for mutation the concentration of the aliquot should be 100µM.\n  * The more mutations specified per construct statistically less colonies will be recovered that have all mutations specified. Assemblies have successfully been recovered with 5 mutations. \n  * The default of number of picked colonies (3) has been validated to recover assemblies with 2-3 mutants. \n[block:callout]\n{\n  \"type\": \"warning\",\n  \"body\": \"It is highly advised that more colonies should be picked if your mutant construct contains more that 2-3 mutations.\",\n  \"title\": \"More colonies should picked if many mutations are being made\"\n}\n[/block]\n  * Variables that affect the price of the run include the number constructs being made, the number of colonies being picked, the number and length of oligonucleotides being synthesized and finally the number of sequencing reactions employed for verification. For this reason the pricing of a run can be fairly dynamic and you will always be shown a quote before you choose to submit the run.\n  * In the event that you are unsuccessful in recovering desired clones from the run, you will currently need to submit another Kunkel run. Plates generated during the Kunkel run cannot undergo re-picking.\n  * For assemblies containing > 2-3 mutation sites/primer, it is highly recommended to increase the ```number of colonies to pick``` parameter under Kunkel \"construct parameters\".\n  * The final samples returned by the Kunkel run are bacterial culture aliquots. Bacterial samples have a shelf life of 3 weeks at 4C.\n  * Downstream of Kunkel mutagenesis purified plasmid may be obtained by performing a Miniprep plasmid purification. This is routinely accomplished successfully by performing the Miniprep within 3 weeks of generating the mutant. The Miniprep protocol can be launched from the protocol browser.\n  * Purified plasmid can be shipped back to your address by following the [return shipment instructions](https://developers.transcriptic.com/docs/return-shipments)\n[block:api-header]\n{\n  \"type\": \"basic\",\n  \"title\": \"Further reading\"\n}\n[/block]\n\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":"kunkel-mutagenesis-1","type":"basic","title":"Kunkel Mutagenesis"}

Kunkel Mutagenesis


[block:callout] { "type": "success", "title": "This is a validated protocol", "body": "The validation document for this protocol is available here http://learn.transcriptic.com/kunkel-mutagenesis/" } [/block] [block:api-header] { "type": "basic", "title": "Description" } [/block] Mutate and generate select DNA plasmids using Kunkel Mutagenesis. This protocol takes a target single stranded DNA molecule and anneals it to a complimentary nucleotide with a base mis-match. DNA polymerase then fills in the missing bases making a double stranded DNA molecule where the antisense strand carries the point mutation. From here DNA replication can take place replicating a sense strand that carries the desired point mutation from the oligonucleotide. 1 or more mutant constructs can be specified with one or more mutations to be made per construct. [block:api-header] { "type": "basic", "title": "Sample requirements" } [/block] [block:parameters] { "data": { "h-0": "Sample", "h-1": "Explanation", "0-1": "A single aliquot of a solution of single stranded DNA. Usually this ssDNA will contain the element (gene, promoter, etc.) you wish to impart a mutation to.", "0-0": "ssDNA", "1-0": "RCA sequencing primers (optional)", "2-0": "Mutant Oligonucleotides (optional)", "1-1": "If sequencing mutant constructs with non-library standard primers they will be required for this protocol.", "2-1": "If you wish to reuse a oligonucleotide to impart a mutation from a previous Kunkel Mutagenesis run, they can be used in future runs." }, "cols": 2, "rows": 3 } [/block] [block:api-header] { "type": "basic", "title": "Required parameters" } [/block] ##Construct Parameters [block:parameters] { "data": { "h-0": "Parameter", "h-1": "Explanation", "0-0": "Number of colonies to pick", "1-0": "Antibiotic resistance", "0-1": "An integer that specifies how many colonies you would like to be picked per mutant generated. Each picked colony will undergo growth and sequencing", "1-1": "Antibiotic to supplement growth media and agar with for colony growth. Choose from Kanamycin, Ampicillin and Spectinomycin" }, "cols": 2, "rows": 2 } [/block] ##Rolling Circle Amplification (RCA) Sequencing Primer(s) [block:callout] { "type": "info", "title": "Choose you primer source", "body": "For sequencing you can choose from stock primers provided by Transcriptic or primers in your inventory." } [/block] [block:parameters] { "data": { "h-0": "Parameter", "h-1": "Explanation", "0-0": "Sequencing Primers", "0-1": "Primers used for sequencing your mutant constructs to confirm the mutation. More than one primer may be specified to sequence multiple regions of the construct. Average read length for the sequencing is approximately 800-900bp." }, "cols": 2, "rows": 1 } [/block] ### Stock Primers Commonly used primers for sequencing MCS regions or other features can be provided by the Transcriptic reagent library. The current stock of primers includes the following list. [block:parameters] { "data": { "h-0": "Primer Name", "0-0": "T7 Forward", "1-0": "T7 Reverse", "2-0": "T3", "3-0": "M13 Reverse", "4-0": "M13 Forward (-20)", "5-0": "M13 Forward (-40)", "6-0": "pGEX Forward", "7-0": "pGEX Reverse", "8-0": "BghRev", "9-0": "ATTL-F", "10-0": "ATTL-R", "11-0": "pET", "12-0": "T7 Forward (short)", "13-0": "gSP6", "14-0": "KBR/TJ", "15-0": "EGFP-C", "16-0": "EGFP-N", "17-0": "CMV-F" }, "cols": 1, "rows": 18 } [/block] ##Mutant Constructs One or more mutant construct can be specified with one or more mutations. To add another mutant construct click the 'Add Mutant' link. [block:callout] { "type": "info", "title": "Specify the mutant constructs desired", "body": "Mutant constructs can specified in 3 ways:\n\n1. Bulk upload of sequences by a `.csv` file\n2. Sequence specified for synthesis during the run\n3. Aliquots from your inventory can be provided. \n\nA CSV template is available for CSV upload from the Protocol Browser." } [/block] ### `.csv` upload Use this functionality if you are specifying many mutant constructs at once. [block:parameters] { "data": { "h-0": "Parameter", "h-1": "Explanation", "0-0": "Mutant constructs", "0-1": "Click 'Upload CSV' to upload a `.csv` file describing your mutant constructs. A template for the CSV file can be downloaded from the Protocol Browser." }, "cols": 2, "rows": 1 } [/block] ### Enter mutant parameters Use this functionality if you are specifying a small number of mutants. [block:parameters] { "data": { "0-0": "Mutant (Group)", "0-1": "Multiple mutants maybe specified, additional mutants can be added by clicking the 'Add Mutant' link at the bottom of the mutant group.\n\nA mutant group contains the fields `Mutant Name` and `Oligonucleotides`.", "1-0": "Mutant Name", "1-1": "The name that you wish to assign to the resulting mutant construct.", "2-0": "Oligonucleotides (Group)", "3-0": "Sequence", "4-0": "Oligo Label", "2-1": "1 or more oligo nucleotides used to insert mutations to a target sequence through a base pair mismatch. To add more oligonucleotides click the 'Add Oligonucleotides' link at the bottom of the Oligonucleotides group.", "3-1": "The sequence of the oligonucleotide that will be used to introduce a mutation.", "4-1": "The label you wish to assign to that oligonucleotide.", "h-0": "Parameter", "h-1": "Explanation" }, "cols": 2, "rows": 5 } [/block] ### Enter Mutant Parameters Selecting Oligos From Your Inventory [block:parameters] { "data": { "h-0": "Parameter", "h-1": "Explanation", "0-0": "Mutant Name", "0-1": "The name you would like to assign to this mutant.", "1-0": "Oligonucleotides", "1-1": "1 or more oligonucleotide aliquots used to insert mutations to a target sequence through a base pair mismatch." }, "cols": 2, "rows": 2 } [/block] [block:api-header] { "type": "basic", "title": "Protocol Outputs" } [/block] [block:parameters] { "data": { "h-0": "Output", "h-1": "Explanation", "0-0": "Plate image", "1-0": "Picked colonies", "2-0": "Sequencing data (optional)", "0-1": "A plate image is returned of all LB-agar cultures for each of the mutants.", "1-1": "The number of colonies picked per well in the LB agar plates is returned.", "2-1": "Data is returned from the Sanger sequencing of the mutant constructs. The sequencing data is returned as a zip file of `.ab1` and `.seq` files" }, "cols": 2, "rows": 3 } [/block] [block:api-header] { "type": "basic", "title": "Recommended guidelines" } [/block] ## ssDNA * ssDNA concentration should be in the range of 30-40ng/µL (the protocol uses 2µL of ssDNA per construct); * ssDNA should be sent to Transcriptic via a sample intake kit and shipped on dry ice and storage of -20C should be specified. no recommendations on length or GC content apart from standard PCR rules since the rxn uses a T7 polymerase. * Typical successful plasmids have been in the size range of ~6.5-7kb * For calculating the volume of ssDNA required per run, the following equation should be used to take into account container dead volumes: ```(number_of_reactions + 3) * 1.5 * 2µL``` For 2 reactions this would be: ```(2 + 3) * 1.5 * 2µL = 15µL``` ## General * Mutation oligonucleotides are recommend to approximately 33 bp for single mutations. Longer oligonucleotides are necessary if they contain more than 1 mutation site. The oligonucleotides should not overlap. If supplying oligonucleotides from your inventory for mutation the concentration of the aliquot should be 100µM. * The more mutations specified per construct statistically less colonies will be recovered that have all mutations specified. Assemblies have successfully been recovered with 5 mutations. * The default of number of picked colonies (3) has been validated to recover assemblies with 2-3 mutants. [block:callout] { "type": "warning", "body": "It is highly advised that more colonies should be picked if your mutant construct contains more that 2-3 mutations.", "title": "More colonies should picked if many mutations are being made" } [/block] * Variables that affect the price of the run include the number constructs being made, the number of colonies being picked, the number and length of oligonucleotides being synthesized and finally the number of sequencing reactions employed for verification. For this reason the pricing of a run can be fairly dynamic and you will always be shown a quote before you choose to submit the run. * In the event that you are unsuccessful in recovering desired clones from the run, you will currently need to submit another Kunkel run. Plates generated during the Kunkel run cannot undergo re-picking. * For assemblies containing > 2-3 mutation sites/primer, it is highly recommended to increase the ```number of colonies to pick``` parameter under Kunkel "construct parameters". * The final samples returned by the Kunkel run are bacterial culture aliquots. Bacterial samples have a shelf life of 3 weeks at 4C. * Downstream of Kunkel mutagenesis purified plasmid may be obtained by performing a Miniprep plasmid purification. This is routinely accomplished successfully by performing the Miniprep within 3 weeks of generating the mutant. The Miniprep protocol can be launched from the protocol browser. * Purified plasmid can be shipped back to your address by following the [return shipment instructions](https://developers.transcriptic.com/docs/return-shipments) [block:api-header] { "type": "basic", "title": "Further reading" } [/block] [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]