{"__v":13,"_id":"568f0ea29ebef90d0087276a","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":"2016-01-08T01:19:30.496Z","link_external":false,"link_url":"","githubsync":"","sync_unique":"","hidden":false,"api":{"results":{"codes":[]},"settings":"","auth":"required","params":[],"url":""},"isReference":false,"order":1,"body":"[block:callout]\n{\n  \"type\": \"success\",\n  \"title\": \"This protocol is validated\",\n  \"body\": \"To view the validation document see [here](http://learn.transcriptic.com/pcr/)\"\n}\n[/block]\n\n[block:api-header]\n{\n  \"type\": \"basic\",\n  \"title\": \"Description\"\n}\n[/block]\nThis protocol will amplify a sequence of DNA provided that selected primers are designed for the target amplicon on the template DNA. The protocol supports generating multiple PCR reactions in a single run. If desired, PCR product can subsequently be purified using the [ExoSAP-IT](doc:exosap-it) protocol.\n[block:api-header]\n{\n  \"type\": \"basic\",\n  \"title\": \"Sample requirements\"\n}\n[/block]\nFor this protocol a minimum or 1 aliquot of template DNA is required in addition to 1 aliquot of appropriate forward primers and 1 aliquot of reverse primers. If performing more than one PCR reaction you must provide a enough sample material to support the number of reactions you specify. It is recommended your template and primer DNA are in aqueous solutions.\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 run you must be sure of the parameters that you provide.\",\n  \"title\": \"Understand your parameters\"\n}\n[/block]\nThe following parameters are required to execute this protocol.\n[block:parameters]\n{\n  \"data\": {\n    \"0-0\": \"DNA volume per reaction\",\n    \"0-1\": \"this is the volume of your template DNA solution that you wish to have in each PCR reaction you do\",\n    \"1-0\": \"Primer volume per reaction\",\n    \"h-0\": \"Parameter\",\n    \"h-1\": \"Explanation\",\n    \"1-1\": \"This is the desired volume of primer solution being added to each reaction vessel.\",\n    \"2-1\": \"You may choose which polymerase to use in your PCR\",\n    \"2-0\": \"Polymerase type\",\n    \"3-1\": \"Your desired total reaction volume for each PCR reaction you are undertaking\",\n    \"3-0\": \"Total reaction volume\",\n    \"4-0\": \"Add MGCL2?\",\n    \"4-1\": \"Check this field if you wish to include 50mM magnesium chloride in your PCR reactions\",\n    \"5-0\": \"MGCL2 volume per reaction\",\n    \"5-1\": \"If you have chosen to include 50 mM magnesium chloride in your PCR reaction, state the volume of magnesium chloride you wish to add to each reaction\",\n    \"6-1\": \"Check this field if you wish to add dimethyl sulfoxide to your PCR reactions\",\n    \"6-0\": \"Add DMSO?\",\n    \"7-1\": \"If you have chosen to add DMSO to your reaction state the volume that you wish to include in each of your PCR reactions\",\n    \"7-0\": \"DMSO volume per reaction\",\n    \"8-1\": \"If you wish to supplement you PCR reactions with any other components you may select a single aliquot here from your inventory to be added to each PCR reaction\",\n    \"8-0\": \"Other additive\",\n    \"9-1\": \"if you have chosen to include an additive to your PCR reactions you must state the volume that you wish each reaction to include\",\n    \"9-0\": \"Volume per reaction\",\n    \"10-1\": \"these parameters are customizable to your experiment if you need additional steps click add step to whichever group you wish to add the step to. You may also add more groups. For each step an incubation `TEMPERATURE` and `TIME` must be specified. For each group of steps the number of cycles must be specified\",\n    \"10-0\": \"Thermocycling parameters\",\n    \"11-1\": \"Check this if you wish for an agarose gel electrophoresis to be run to analyse your reaction products\",\n    \"11-0\": \"Run a gel?\",\n    \"12-1\": \"if you have chosen to run a gel, specify the volume of reaction product you wish to be added to a lane in the gel\",\n    \"12-0\": \"Volume of reaction to run\",\n    \"13-1\": \"Depending on your expected PCR products specify a relevant ladder choice.\\n\\n`Low range` has markers at 100bp, 250bp, 500bp, 1000bp, 2000bp\\n\\n`High range` has markers at 500bp, 1kbp, 2kbp, 4kbp, 10kbp\\n\\nMore information on ladders can be found at [Gel Electrophoresis](doc:gel-electrophoresis).\",\n    \"13-0\": \"Ladder\",\n    \"14-1\": \"specify the concentration of agarose in the gel being used. Remember for shorter lengths of DNA high concentrations of agarose achieve better separation\",\n    \"14-0\": \"Agarose concentration (mass/volume %)\"\n  },\n  \"cols\": 2,\n  \"rows\": 15\n}\n[/block]\n\n[block:api-header]\n{\n  \"type\": \"basic\",\n  \"title\": \"Protocol Outputs\"\n}\n[/block]\nEach PCR reaction will result in a new container being added to your inventory containing the result of your PCR reaction. Furthermore your template DNA and primer DNA containers will be returned to the inventory in addition to any custom additive you may have specified.\n\nIf you have specified that an agarose gel should be run, an image file will also be generated showing your gel.\n[block:api-header]\n{\n  \"type\": \"basic\",\n  \"title\": \"Recommended guidelines\"\n}\n[/block]\n## PCR\n\nPCR is highly dependent on the polymerase you use in addition to your template and primer DNA. It is essential that you have properly designed your primers for your target amplicon. A rough guide is that your final quantity of template DNA in your PCR reaction should be <250ng. \n\nFor your primers it is recommended you keep them at a stock concentration of 10µM in your inventory. Then for your PCR reactions you will require approximately 1µL of 10mM primer for a 20µL reaction or 2.5µL for a 50µL reaction. Be sure to use both forward and reverse primers in your PCR reactions.\n\nIt is recommended that you follow the vendor guidelines on PCR for whichever DNA polymerase you choose to use. These will inform you of the correct concentrations of template DNA and primer DNA that you should include in your PCR reactions.\n\nThe supplementation of PCR reactions with magnesium chloride and DMSO may be required for difficult to replicate templates, you should follow the manufacturers guidelines for your DNA polymerase of choice.\n\n\n## Agarose Gel Electrophoresis\n\nTo achieve a successful agarose gel electrophoresis separation of your PCR product ensure you are using a suitable agarose percentage in your gel. Below is a table of suitable DNA size ranges alongside the 3 choices of agarose gel % available in the protocol.\n\n|**Agarose Percentage (%)**|**DNA size range (bp)**|\n|-----|------|\n|2| 50 - 2,000|\n|1.2|400 - 7,000|\n|0.8|800 - 12,000|\n[block:api-header]\n{\n  \"type\": \"basic\",\n  \"title\": \"Further reading\"\n}\n[/block]\n[Phusion DNA polymerase guidelines](https://www.neb.com/products/m0530-phusion-high-fidelity-dna-polymerase#tabselect2)\n[MyTaq DNA polymerase further information](http://www.bioline.com/us/mytaq-hs-dna-polymerase.html)\n[KAPA2G Fast Hot Start DNA polymerase further information](https://www.kapabiosystems.com/product-applications/products/pcr-2/kapa2g-fast-pcr-kits/)\n[KAPA HIFI DNA polymerase](https://www.kapabiosystems.com/product-applications/products/pcr-2/kapa-hifi-pcr-kits/)\n[block:callout]\n{\n  \"type\": \"success\",\n  \"body\": \"For further advice on executing the PCR 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":"pcr","type":"basic","title":"BasicPCR"}
[block:callout] { "type": "success", "title": "This protocol is validated", "body": "To view the validation document see [here](http://learn.transcriptic.com/pcr/)" } [/block] [block:api-header] { "type": "basic", "title": "Description" } [/block] This protocol will amplify a sequence of DNA provided that selected primers are designed for the target amplicon on the template DNA. The protocol supports generating multiple PCR reactions in a single run. If desired, PCR product can subsequently be purified using the [ExoSAP-IT](doc:exosap-it) protocol. [block:api-header] { "type": "basic", "title": "Sample requirements" } [/block] For this protocol a minimum or 1 aliquot of template DNA is required in addition to 1 aliquot of appropriate forward primers and 1 aliquot of reverse primers. If performing more than one PCR reaction you must provide a enough sample material to support the number of reactions you specify. It is recommended your template and primer DNA are in aqueous solutions. [block:api-header] { "type": "basic", "title": "Required parameters" } [/block] [block:callout] { "type": "warning", "body": "For you to successfully execute a PCR run you must be sure of the parameters that you provide.", "title": "Understand your parameters" } [/block] The following parameters are required to execute this protocol. [block:parameters] { "data": { "0-0": "DNA volume per reaction", "0-1": "this is the volume of your template DNA solution that you wish to have in each PCR reaction you do", "1-0": "Primer volume per reaction", "h-0": "Parameter", "h-1": "Explanation", "1-1": "This is the desired volume of primer solution being added to each reaction vessel.", "2-1": "You may choose which polymerase to use in your PCR", "2-0": "Polymerase type", "3-1": "Your desired total reaction volume for each PCR reaction you are undertaking", "3-0": "Total reaction volume", "4-0": "Add MGCL2?", "4-1": "Check this field if you wish to include 50mM magnesium chloride in your PCR reactions", "5-0": "MGCL2 volume per reaction", "5-1": "If you have chosen to include 50 mM magnesium chloride in your PCR reaction, state the volume of magnesium chloride you wish to add to each reaction", "6-1": "Check this field if you wish to add dimethyl sulfoxide to your PCR reactions", "6-0": "Add DMSO?", "7-1": "If you have chosen to add DMSO to your reaction state the volume that you wish to include in each of your PCR reactions", "7-0": "DMSO volume per reaction", "8-1": "If you wish to supplement you PCR reactions with any other components you may select a single aliquot here from your inventory to be added to each PCR reaction", "8-0": "Other additive", "9-1": "if you have chosen to include an additive to your PCR reactions you must state the volume that you wish each reaction to include", "9-0": "Volume per reaction", "10-1": "these parameters are customizable to your experiment if you need additional steps click add step to whichever group you wish to add the step to. You may also add more groups. For each step an incubation `TEMPERATURE` and `TIME` must be specified. For each group of steps the number of cycles must be specified", "10-0": "Thermocycling parameters", "11-1": "Check this if you wish for an agarose gel electrophoresis to be run to analyse your reaction products", "11-0": "Run a gel?", "12-1": "if you have chosen to run a gel, specify the volume of reaction product you wish to be added to a lane in the gel", "12-0": "Volume of reaction to run", "13-1": "Depending on your expected PCR products specify a relevant ladder choice.\n\n`Low range` has markers at 100bp, 250bp, 500bp, 1000bp, 2000bp\n\n`High range` has markers at 500bp, 1kbp, 2kbp, 4kbp, 10kbp\n\nMore information on ladders can be found at [Gel Electrophoresis](doc:gel-electrophoresis).", "13-0": "Ladder", "14-1": "specify the concentration of agarose in the gel being used. Remember for shorter lengths of DNA high concentrations of agarose achieve better separation", "14-0": "Agarose concentration (mass/volume %)" }, "cols": 2, "rows": 15 } [/block] [block:api-header] { "type": "basic", "title": "Protocol Outputs" } [/block] Each PCR reaction will result in a new container being added to your inventory containing the result of your PCR reaction. Furthermore your template DNA and primer DNA containers will be returned to the inventory in addition to any custom additive you may have specified. If you have specified that an agarose gel should be run, an image file will also be generated showing your gel. [block:api-header] { "type": "basic", "title": "Recommended guidelines" } [/block] ## PCR PCR is highly dependent on the polymerase you use in addition to your template and primer DNA. It is essential that you have properly designed your primers for your target amplicon. A rough guide is that your final quantity of template DNA in your PCR reaction should be <250ng. For your primers it is recommended you keep them at a stock concentration of 10µM in your inventory. Then for your PCR reactions you will require approximately 1µL of 10mM primer for a 20µL reaction or 2.5µL for a 50µL reaction. Be sure to use both forward and reverse primers in your PCR reactions. It is recommended that you follow the vendor guidelines on PCR for whichever DNA polymerase you choose to use. These will inform you of the correct concentrations of template DNA and primer DNA that you should include in your PCR reactions. The supplementation of PCR reactions with magnesium chloride and DMSO may be required for difficult to replicate templates, you should follow the manufacturers guidelines for your DNA polymerase of choice. ## Agarose Gel Electrophoresis To achieve a successful agarose gel electrophoresis separation of your PCR product ensure you are using a suitable agarose percentage in your gel. Below is a table of suitable DNA size ranges alongside the 3 choices of agarose gel % available in the protocol. |**Agarose Percentage (%)**|**DNA size range (bp)**| |-----|------| |2| 50 - 2,000| |1.2|400 - 7,000| |0.8|800 - 12,000| [block:api-header] { "type": "basic", "title": "Further reading" } [/block] [Phusion DNA polymerase guidelines](https://www.neb.com/products/m0530-phusion-high-fidelity-dna-polymerase#tabselect2) [MyTaq DNA polymerase further information](http://www.bioline.com/us/mytaq-hs-dna-polymerase.html) [KAPA2G Fast Hot Start DNA polymerase further information](https://www.kapabiosystems.com/product-applications/products/pcr-2/kapa2g-fast-pcr-kits/) [KAPA HIFI DNA polymerase](https://www.kapabiosystems.com/product-applications/products/pcr-2/kapa-hifi-pcr-kits/) [block:callout] { "type": "success", "body": "For further advice on executing the PCR protocol please ask questions to the community at https://forum.transcriptic.com", "title": "Check out the forum if you have more questions." } [/block]