mirror of
https://github.com/scinote-eln/scinote-web.git
synced 2024-11-17 14:46:00 +08:00
1 line
No EOL
27 KiB
JSON
1 line
No EOL
27 KiB
JSON
{"experiment":{"id":433,"name":"Development of SNP markers panel","description":"This template shows how to generate a SNP markers panel with the preparation of SNP panel.\r\nSingle nucleotide polymorphisms (SNPs) markers are widely used for genomic research and breeding. Development of a panel of diagnostic SNP markers is used for the genotyping as well as the identification of hybrids and interspecies introgression events.","project_id":223,"created_by_id":202,"last_modified_by_id":202,"archived":false,"archived_by_id":null,"archived_on":null,"restored_by_id":null,"restored_on":null,"created_at":"2018-11-12T08:12:09.669Z","updated_at":"2019-02-07T14:44:00.337Z","workflowimg_file_name":"wimg20181217-1-jr6hrp.png","workflowimg_content_type":"image/png","workflowimg_file_size":1486,"workflowimg_updated_at":"2019-02-07T14:44:00.337Z","uuid":"de7f0d1b-f842-440b-a367-a0333a11430c"},"my_modules":[{"my_module":{"id":2150,"name":"RAD library preparation and sequencing","due_date":null,"description":null,"x":34,"y":0,"my_module_group_id":1167,"created_at":"2018-11-12T08:39:44.221Z","updated_at":"2018-12-17T07:51:25.145Z","archived":false,"archived_on":null,"created_by_id":202,"last_modified_by_id":202,"archived_by_id":null,"restored_by_id":null,"restored_on":null,"nr_of_assigned_samples":0,"workflow_order":1,"experiment_id":433,"state":"uncompleted","completed_on":null},"outputs":[{"id":5924,"input_id":2151,"output_id":2150}],"my_module_tags":[],"task_comments":[],"my_module_repository_rows":[],"user_my_modules":[],"protocols":[{"protocol":{"id":3488,"name":null,"authors":null,"description":null,"added_by_id":null,"my_module_id":2150,"team_id":1,"protocol_type":"unlinked","parent_id":null,"parent_updated_at":null,"archived_by_id":null,"archived_on":null,"restored_by_id":null,"restored_on":null,"created_at":"2018-11-12T08:39:44.225Z","updated_at":"2019-02-20T07:58:51.770Z","published_on":null,"nr_of_linked_children":0},"protocol_protocol_keywords":[],"steps":[{"step":{"id":5192,"name":"PCR genotyping","description":"\u003c!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\"\u003e\n\u003chtml\u003e\u003cbody\u003e\n\u003cp\u003ePCR conditions: \u003c/p\u003e\n\u003cul\u003e\n\u003cli\u003e95°C for 1 min\u003c/li\u003e\n\u003cli\u003e35 cycles: 95°C for 15 s\u003c/li\u003e\n\u003cli\u003e56°C for 15 s\u003c/li\u003e\n\u003cli\u003e72°C for 22 s\u003c/li\u003e\n\u003c/ul\u003e\n\u003cbr\u003ePCR products run at \u003cstrong\u003e60 V\u003c/strong\u003e for \u003cstrong\u003e40 mins\u003c/strong\u003e on a\u003cstrong\u003e 2%\u003c/strong\u003e agarose gel (\u003cstrong\u003e0.5× TAE\u003c/strong\u003e, stained with \u003cstrong\u003e100 ng/μL\u003c/strong\u003e EtBr).\u003c/body\u003e\u003c/html\u003e","position":0,"completed":false,"completed_on":null,"user_id":202,"created_at":"2018-12-17T07:34:16.845Z","updated_at":"2019-02-19T14:35:53.498Z","last_modified_by_id":202,"protocol_id":3488},"checklists":[],"step_comments":[],"step_assets":[],"assets":[],"step_tables":[{"id":531,"step_id":5192,"table_id":668}],"tables":[{"id":668,"created_at":"2018-12-17T07:35:02.552Z","updated_at":"2019-02-19T14:35:53.484Z","created_by_id":202,"last_modified_by_id":202,"name":"PCR Mastermix","team_id":1,"contents":"eyJkYXRhIjpbWyJDb21wb25lbnQiLCJWb2x1bWUgKMK1bCkiLCJGaW5hbCBD\nb25jLiJdLFsiTXlUYXEgbWl4IiwiMyIsIjF4Il0sWyJGb3J3YXJkIHByaW1l\nciIsIjAsNCIsIjEwIM68TSJdLFsiUmV2ZXJzZSBwcmltZXIiLCIwLDQiLCIx\nMCDOvE0iXSxbIlRlbXBsYXRlIEROQSIsIjAsNSIsIjXigJM1MCBuZy/OvEwi\nXSxbIlVsdHJhcHVyZSB3YXRlciIsIjEsNyIsIiJdXX0=\n","data_vector":"JzAnOjEzLDIwLDI3ICcxJzozOCAnMTAnOjE1LDIyICcxeCc6MTAgJzIwMCc6\nMzEgJzIyMzUwJzozMiAnMjY1bCc6NCAnMjc0bCc6MzUgJzI3NG0nOjE3LDI0\nICczJzo5ICczMDInOjMgJzMxNic6MTYsMjMsMzQgJzM0Mic6MzAgJzQnOjE0\nLDIxICc1JzoyOCwyOSAnNyc6MzkgJ2NvbXBvbmVudCc6MSAnY29uYyc6NiAn\nZG5hJzoyNiAnZmluYWwnOjUgJ2ZvcndhcmQnOjExICdtaXgnOjggJ215dGFx\nJzo3ICduZyc6MzMgJ3ByaW1lcic6MTIsMTkgJ3JldmVyc2UnOjE4ICd0ZW1w\nbGF0ZSc6MjUgJ3VsdHJhcHVyZSc6MzYgJ3ZvbHVtZSc6MiAnd2F0ZXInOjM3\n"}]},{"step":{"id":4422,"name":"Shearing and size selection","description":"\u003c!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\"\u003e\n\u003chtml\u003e\u003cbody\u003e\u003cp\u003eLibraries can be quantified by fluorimetry and calibrated by sequencing. \u003c/p\u003e\u003c/body\u003e\u003c/html\u003e","position":2,"completed":false,"completed_on":null,"user_id":202,"created_at":"2018-11-12T14:41:18.099Z","updated_at":"2019-02-20T07:37:25.765Z","last_modified_by_id":202,"protocol_id":3488},"checklists":[{"checklist":{"id":960,"name":"Guidelines:","step_id":4422,"created_at":"2018-12-21T10:22:55.963Z","updated_at":"2018-12-21T10:22:55.963Z","created_by_id":null,"last_modified_by_id":null},"checklist_items":[{"id":3950,"text":"Size selection (100 to 800 bp) by agarose gel electrophoresis.","checked":false,"checklist_id":960,"created_at":"2018-12-21T10:22:55.965Z","updated_at":"2018-12-21T10:22:55.965Z","created_by_id":null,"last_modified_by_id":null,"position":0},{"id":3951,"text":"Gel purification, end repair, dA overhang addition, P2 paired-end adapter ligation and library amplification.","checked":false,"checklist_id":960,"created_at":"2018-12-21T10:22:55.972Z","updated_at":"2018-12-21T10:22:55.972Z","created_by_id":null,"last_modified_by_id":null,"position":1},{"id":3952,"text":"120 μL of each amplified library is size-selected (about 250 to 500 bp) by gel electrophoresis.","checked":false,"checklist_id":960,"created_at":"2018-12-21T10:22:55.981Z","updated_at":"2018-12-21T10:22:55.981Z","created_by_id":null,"last_modified_by_id":null,"position":2},{"id":3953,"text":"Final libraries are put through quality control and high-throughput sequencing.","checked":false,"checklist_id":960,"created_at":"2018-12-21T10:22:55.989Z","updated_at":"2018-12-21T10:23:18.774Z","created_by_id":null,"last_modified_by_id":null,"position":3}]}],"step_comments":[],"step_assets":[{"id":3084,"step_id":4422,"asset_id":3746},{"id":3085,"step_id":4422,"asset_id":3747}],"assets":[{"id":3746,"created_at":"2019-02-20T07:36:46.773Z","updated_at":"2019-02-20T07:37:25.383Z","file_file_name":"Shearing.jpg","file_content_type":"image/jpeg","file_file_size":17969,"file_updated_at":"2019-02-20T07:37:24.952Z","created_by_id":202,"last_modified_by_id":null,"estimated_size":19765,"file_present":true,"lock":null,"lock_ttl":null,"version":1,"file_processing":false,"team_id":1},{"id":3747,"created_at":"2019-02-20T07:36:46.946Z","updated_at":"2019-02-20T07:37:25.757Z","file_file_name":"Amplification.jpg","file_content_type":"image/jpeg","file_file_size":15037,"file_updated_at":"2019-02-20T07:37:25.431Z","created_by_id":202,"last_modified_by_id":null,"estimated_size":16540,"file_present":true,"lock":null,"lock_ttl":null,"version":1,"file_processing":false,"team_id":1}],"step_tables":[],"tables":[]},{"step":{"id":4420,"name":"RAD library preparation","description":"\u003c!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\"\u003e\n\u003chtml\u003e\u003cbody\u003e\n\u003cp\u003e The RAD library was prepared as originally described in \u003ca href=\"https://www.ncbi.nlm.nih.gov/pubmed/18852878/\" target=\"_blank\" rel=\"noopener\"\u003earticle\u003c/a\u003e.\u003cbr\u003e\u003cbr\u003eAt least \u003cstrong\u003e30\u003c/strong\u003e specimens need to be chosen.\u003cbr\u003e\u003cbr\u003e\u003cstrong\u003eEach sample is digested at 37°C for 40 min with restriction enzyme:\u003c/strong\u003e\u003c/p\u003e\n\u003cul\u003e\n\u003cli\u003e\n\u003cstrong\u003e6 U\u003c/strong\u003e \u003cem\u003erestriction enzyme\u003c/em\u003e per μg genomic DNA in \u003cstrong\u003e1×\u003c/strong\u003e Reaction Buffer 4 at a final concentration of about \u003cstrong\u003e1 μg\u003c/strong\u003e DNA per \u003cstrong\u003e50 μL\u003c/strong\u003e reaction volume\u003c/li\u003e\n\u003cli\u003eThe samples are heat-inactivated at \u003cstrong\u003e65°C\u003c/strong\u003e for \u003cstrong\u003e20 min\u003c/strong\u003e.\u003c/li\u003e\n\u003c/ul\u003e\n\u003cbr\u003e\u003cstrong\u003eIndividual-specific P1 adapters, each with a unique 5 or 7 bp barcode is ligated to the digested DNA at 22°C for 15 min by adding:\u003c/strong\u003e\u003cbr\u003e\u003cbr\u003e\n\u003cul\u003e\n\u003cli\u003e0.6 μL (DNA samples) 100 nmol/L P1 adapter\u003c/li\u003e\n\u003cli\u003e0.15 μL 100 mmol/L rATP \u003c/li\u003e\n\u003cli\u003e0.25 μL 10× Reaction Buffer 2\u003c/li\u003e\n\u003cli\u003e0.125 μL T4 ligase \u003c/li\u003e\n\u003cli\u003ereaction volumes made up to 15 μL with nuclease-free water \u003c/li\u003e\n\u003c/ul\u003e\n\u003cbr\u003eAfter heat-inactivation, the ligation reactions are slowly cooled to room temperature then combined in appropriate multiplex pools.\u003c/body\u003e\u003c/html\u003e","position":1,"completed":false,"completed_on":null,"user_id":202,"created_at":"2018-11-12T14:34:29.463Z","updated_at":"2019-02-20T07:58:51.732Z","last_modified_by_id":202,"protocol_id":3488},"checklists":[],"step_comments":[],"step_assets":[{"id":3083,"step_id":4420,"asset_id":3745}],"assets":[{"id":3745,"created_at":"2019-02-20T07:36:16.089Z","updated_at":"2019-02-20T07:58:51.655Z","file_file_name":"Digestion_of_DNA.jpg","file_content_type":"image/jpeg","file_file_size":14817,"file_updated_at":"2019-02-20T07:36:24.506Z","created_by_id":202,"last_modified_by_id":202,"estimated_size":16298,"file_present":true,"lock":null,"lock_ttl":null,"version":1,"file_processing":false,"team_id":1}],"step_tables":[],"tables":[]}]}],"results":[]},{"my_module":{"id":2151,"name":"Genotyping RAD alleles","due_date":null,"description":null,"x":68,"y":0,"my_module_group_id":1167,"created_at":"2018-11-12T08:39:44.243Z","updated_at":"2018-12-17T07:51:25.147Z","archived":false,"archived_on":null,"created_by_id":202,"last_modified_by_id":202,"archived_by_id":null,"restored_by_id":null,"restored_on":null,"nr_of_assigned_samples":0,"workflow_order":2,"experiment_id":433,"state":"uncompleted","completed_on":null},"outputs":[{"id":5925,"input_id":2154,"output_id":2151}],"my_module_tags":[],"task_comments":[],"my_module_repository_rows":[],"user_my_modules":[],"protocols":[{"protocol":{"id":3489,"name":null,"authors":null,"description":null,"added_by_id":null,"my_module_id":2151,"team_id":1,"protocol_type":"unlinked","parent_id":null,"parent_updated_at":null,"archived_by_id":null,"archived_on":null,"restored_by_id":null,"restored_on":null,"created_at":"2018-11-12T08:39:44.247Z","updated_at":"2018-12-21T10:26:41.366Z","published_on":null,"nr_of_linked_children":0},"protocol_protocol_keywords":[],"steps":[{"step":{"id":4424,"name":"RAD markers","description":"\u003c!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\"\u003e\n\u003chtml\u003e\u003cbody\u003e\n\u003cp\u003eReads of low quality must be discarded. \u003cbr\u003eRetained reads are sorted into loci and genotypes using software developed for that purpose. It will assigns loci using a likelihood-based algorithm to separate actual SNPs from SNPs likely to have arisen from sequencing error. \u003cbr\u003e\u003cbr\u003e\u003cstrong\u003eParameters:\u003c/strong\u003e\u003cbr\u003e\n\u003c/p\u003e\n\u003cul\u003e\n\u003cli\u003eDe novo assembly parameters.\u003c/li\u003e\n\u003cli\u003eMinimum stack depth of 5.\u003c/li\u003e\n\u003cli\u003eMaximum of 2 mismatches.\u003c/li\u003e\n\u003cli\u003eNo more than 1 mismatch between alleles.\u003c/li\u003e\n\u003c/ul\u003e\n\u003c/body\u003e\u003c/html\u003e","position":0,"completed":false,"completed_on":null,"user_id":202,"created_at":"2018-11-12T14:59:18.147Z","updated_at":"2018-12-21T10:25:44.853Z","last_modified_by_id":202,"protocol_id":3489},"checklists":[],"step_comments":[],"step_assets":[],"assets":[],"step_tables":[],"tables":[]},{"step":{"id":5194,"name":"RAD library construction","description":"\u003c!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\"\u003e\n\u003chtml\u003e\u003cbody\u003e\u003cp\u003eSequencing data from filtered Informative RAD markers is combined into a single alignment of alleles in RAD library construction. Data analysis is carried out using R and an associated R/\u003cem\u003eadegenet\u003c/em\u003e package for Principal Component Analysis (PCA) and Discriminant Analysis of Principal Components (DAPC).\u003cem\u003e\u003cstrong\u003e PCA creates simplified models of the total variation within the dataset.\u003c/strong\u003e \u003cstrong\u003eDAPC identifies clusters of genetically related individuals.\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\u003c/body\u003e\u003c/html\u003e","position":1,"completed":false,"completed_on":null,"user_id":202,"created_at":"2018-12-17T07:50:46.898Z","updated_at":"2018-12-21T10:26:41.233Z","last_modified_by_id":202,"protocol_id":3489},"checklists":[],"step_comments":[],"step_assets":[],"assets":[],"step_tables":[],"tables":[]}]}],"results":[]},{"my_module":{"id":2154,"name":"SNP-assay design","due_date":null,"description":null,"x":102,"y":0,"my_module_group_id":1167,"created_at":"2018-11-12T08:39:44.312Z","updated_at":"2018-12-17T07:51:25.150Z","archived":false,"archived_on":null,"created_by_id":202,"last_modified_by_id":202,"archived_by_id":null,"restored_by_id":null,"restored_on":null,"nr_of_assigned_samples":0,"workflow_order":3,"experiment_id":433,"state":"uncompleted","completed_on":null},"outputs":[],"my_module_tags":[],"task_comments":[],"my_module_repository_rows":[],"user_my_modules":[],"protocols":[{"protocol":{"id":3492,"name":null,"authors":null,"description":null,"added_by_id":null,"my_module_id":2154,"team_id":1,"protocol_type":"unlinked","parent_id":null,"parent_updated_at":null,"archived_by_id":null,"archived_on":null,"restored_by_id":null,"restored_on":null,"created_at":"2018-11-12T08:39:44.325Z","updated_at":"2019-02-20T07:37:09.688Z","published_on":null,"nr_of_linked_children":0},"protocol_protocol_keywords":[],"steps":[{"step":{"id":4428,"name":"Genotype assignment","description":"\u003c!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\"\u003e\n\u003chtml\u003e\u003cbody\u003e\u003cp\u003eReading the fluorescence emission of the FAM and HEX fluorophores for each sample, in comparison to no-template control reactions, using a Real Time PCR Thermal Cycler and Quansoft endpoint genotyping software.\u003c/p\u003e\u003c/body\u003e\u003c/html\u003e","position":1,"completed":false,"completed_on":null,"user_id":202,"created_at":"2018-11-12T15:22:18.606Z","updated_at":"2018-11-12T15:22:18.606Z","last_modified_by_id":202,"protocol_id":3492},"checklists":[],"step_comments":[],"step_assets":[],"assets":[],"step_tables":[],"tables":[]},{"step":{"id":4427,"name":"SNP assays","description":"\u003c!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\"\u003e\n\u003chtml\u003e\u003cbody\u003e\n\u003cp\u003eSNP of interest at a given locus need to be at least \u003cstrong\u003e20 bp\u003c/strong\u003e from the end of a given sequence, to allow for primer design. SNP assays were designed and manufactured for use with KASP genotyping technology.\u003cbr\u003e\u003cbr\u003e\u003cstrong\u003eOptimisation assay conditions:\u003c/strong\u003e\u003c/p\u003e\n\u003cul\u003e\n\u003cli\u003e94°C for 15 min;\u003c/li\u003e\n\u003cli\u003e10 cycles 94°C for 20 s, 61–55°C for 120 s (0.6°C drop per cycle)\u003c/li\u003e\n\u003cli\u003e40 cycles 94°C for 20 s, 55°C for 120 s \u003c/li\u003e\n\u003c/ul\u003e\n\u003c/body\u003e\u003c/html\u003e","position":0,"completed":false,"completed_on":null,"user_id":202,"created_at":"2018-11-12T15:20:06.636Z","updated_at":"2019-02-20T07:37:26.227Z","last_modified_by_id":202,"protocol_id":3492},"checklists":[],"step_comments":[],"step_assets":[{"id":3086,"step_id":4427,"asset_id":3748}],"assets":[{"id":3748,"created_at":"2019-02-20T07:37:09.362Z","updated_at":"2019-02-20T07:37:26.219Z","file_file_name":"SNP_marker.jpg","file_content_type":"image/jpeg","file_file_size":21886,"file_updated_at":"2019-02-20T07:37:25.800Z","created_by_id":202,"last_modified_by_id":null,"estimated_size":24074,"file_present":true,"lock":null,"lock_ttl":null,"version":1,"file_processing":false,"team_id":1}],"step_tables":[{"id":455,"step_id":4427,"table_id":573}],"tables":[{"id":573,"created_at":"2018-11-12T15:20:07.148Z","updated_at":"2019-02-20T07:37:09.513Z","created_by_id":202,"last_modified_by_id":202,"name":"Reaction mastermix","team_id":1,"contents":"eyJkYXRhIjpbWyJDb21wb25lbnQiLCJWb2x1bWUiXSxbIjLDlyBLQVNQIE1h\nc3RlciBNaXgiLCIyLjUgzrxMIl0sWyJLQVNQIEFzc2F5IE1peCIsIjAuMDcg\nzrxMIl0sWyJUZW1wbGF0ZSBETkEiLCIwLjQgzrxMIl0sWyJVbHRyYXB1cmUg\nd2F0ZXIiLCIyLjEgzrxMIl1dfQ==\n","data_vector":"JzAuMDcnOjE1ICcwLjQnOjIwICcyJzozICcyLjEnOjI1ICcyLjUnOjkgJzIy\nNyc6NSAnMjc0bCc6MTEsMTcsMjIsMjcgJzMwMyc6NCAnMzE2JzoxMCwxNiwy\nMSwyNiAnYXNzYXknOjEzICdjb21wb25lbnQnOjEgJ2RuYSc6MTkgJ2thc3An\nOjYsMTIgJ21hc3Rlcic6NyAnbWl4Jzo4LDE0ICd0ZW1wbGF0ZSc6MTggJ3Vs\ndHJhcHVyZSc6MjMgJ3ZvbHVtZSc6MiAnd2F0ZXInOjI0\n"}]}]}],"results":[]},{"my_module":{"id":2149,"name":"DNA extracting","due_date":null,"description":null,"x":0,"y":0,"my_module_group_id":1167,"created_at":"2018-11-12T08:39:44.191Z","updated_at":"2018-12-17T07:51:25.152Z","archived":false,"archived_on":null,"created_by_id":202,"last_modified_by_id":202,"archived_by_id":null,"restored_by_id":null,"restored_on":null,"nr_of_assigned_samples":0,"workflow_order":0,"experiment_id":433,"state":"uncompleted","completed_on":null},"outputs":[{"id":5923,"input_id":2150,"output_id":2149}],"my_module_tags":[],"task_comments":[],"my_module_repository_rows":[],"user_my_modules":[],"protocols":[{"protocol":{"id":3487,"name":null,"authors":null,"description":null,"added_by_id":null,"my_module_id":2149,"team_id":1,"protocol_type":"unlinked","parent_id":null,"parent_updated_at":null,"archived_by_id":null,"archived_on":null,"restored_by_id":null,"restored_on":null,"created_at":"2018-11-12T08:39:44.202Z","updated_at":"2019-02-19T14:34:47.554Z","published_on":null,"nr_of_linked_children":0},"protocol_protocol_keywords":[],"steps":[{"step":{"id":4416,"name":"Protein extraction and RNAse treatment","description":"\u003c!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\"\u003e\n\u003chtml\u003e\u003cbody\u003e\u003cp\u003eHere are the guidelines for protein extraction: \u003c/p\u003e\u003c/body\u003e\u003c/html\u003e","position":1,"completed":false,"completed_on":null,"user_id":202,"created_at":"2018-11-12T12:00:57.856Z","updated_at":"2018-12-24T08:46:24.129Z","last_modified_by_id":202,"protocol_id":3487},"checklists":[{"checklist":{"id":958,"name":"Guidelines:","step_id":4416,"created_at":"2018-12-21T09:50:59.139Z","updated_at":"2018-12-21T09:50:59.139Z","created_by_id":null,"last_modified_by_id":null},"checklist_items":[{"id":3939,"text":"Add 1 volume of chloroform: Isoamyl alcohol to the solution and mix by inversion for 5 min. Centrifuge the sample for 10 min at 5000 × g and pipette the upper aqueous phase into a new Falcon tube.","checked":false,"checklist_id":958,"created_at":"2018-12-21T09:50:59.141Z","updated_at":"2018-12-21T09:50:59.141Z","created_by_id":null,"last_modified_by_id":null,"position":0},{"id":3940,"text":"Add 5 μL of RNAse A to the solution and incubate at 37°C for 15 min with periodic, gentle mixing.","checked":false,"checklist_id":958,"created_at":"2018-12-21T09:50:59.149Z","updated_at":"2018-12-21T09:50:59.149Z","created_by_id":null,"last_modified_by_id":null,"position":1},{"id":3941,"text":"After incubation, add 1 volume of chloroform: Isoamyl alcohol to the solution and mix by inversion for 5 min.","checked":false,"checklist_id":958,"created_at":"2018-12-21T09:50:59.156Z","updated_at":"2018-12-21T09:50:59.156Z","created_by_id":null,"last_modified_by_id":null,"position":2},{"id":3942,"text":"Centrifuge the solution for 10 min at 5000 × g and pipette the aqueous phase into a new Falcon tube.","checked":false,"checklist_id":958,"created_at":"2018-12-21T09:50:59.163Z","updated_at":"2018-12-21T09:50:59.163Z","created_by_id":null,"last_modified_by_id":null,"position":3}]}],"step_comments":[],"step_assets":[],"assets":[],"step_tables":[],"tables":[]},{"step":{"id":4418,"name":"DNA quality and quantity assessment","description":"\u003c!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\"\u003e\n\u003chtml\u003e\u003cbody\u003e\n\u003cp\u003eLooking for a single absorbance peak at \u003cstrong\u003e260 nm\u003c/strong\u003e, a \u003cstrong\u003e260/280\u003c/strong\u003e absorbance ratio of \u003cstrong\u003e1.8-2.0\u003c/strong\u003e and no evidence of substantial band shearing or contamination (either RNA or polysaccharide).\u003cbr\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAssess the quality of the extracted DNA:\u003c/strong\u003e\u003c/p\u003e\n\u003col\u003e\n\u003cli\u003eNanoDrop UV/Vis spectrophotometer\u003c/li\u003e\n\u003cli\u003e0.7% (w/v) agarose gel electrophoresis\u003c/li\u003e\n\u003c/ol\u003e\n\u003c/body\u003e\u003c/html\u003e","position":3,"completed":false,"completed_on":null,"user_id":202,"created_at":"2018-11-12T12:22:47.725Z","updated_at":"2019-02-19T14:34:47.503Z","last_modified_by_id":202,"protocol_id":3487},"checklists":[],"step_comments":[],"step_assets":[],"assets":[],"step_tables":[],"tables":[]},{"step":{"id":4414,"name":"Preparation","description":"\u003c!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\"\u003e\n\u003chtml\u003e\u003cbody\u003e\u003cp\u003eGuidelines for samples preparation bellow. \u003c/p\u003e\u003c/body\u003e\u003c/html\u003e","position":0,"completed":false,"completed_on":null,"user_id":202,"created_at":"2018-11-12T11:06:48.312Z","updated_at":"2018-12-24T08:34:20.404Z","last_modified_by_id":202,"protocol_id":3487},"checklists":[{"checklist":{"id":957,"name":"Guidelines","step_id":4414,"created_at":"2018-12-21T09:43:55.386Z","updated_at":"2018-12-21T09:43:55.386Z","created_by_id":null,"last_modified_by_id":null},"checklist_items":[{"id":3934,"text":"Samples are stored in 95% ethanol solution at -20°C.","checked":false,"checklist_id":957,"created_at":"2018-12-21T09:43:55.389Z","updated_at":"2018-12-21T09:43:55.389Z","created_by_id":null,"last_modified_by_id":null,"position":0},{"id":3935,"text":"Pre-heat water baths (65°C and 37°C).","checked":false,"checklist_id":957,"created_at":"2018-12-21T09:43:55.396Z","updated_at":"2018-12-21T09:43:55.396Z","created_by_id":null,"last_modified_by_id":null,"position":1},{"id":3936,"text":"Prepare 10 mL (per 1 g of sample) extraction buffer by adding 0.3% (v/v) β-mercaptoethanol in a 50 mL Falcon tube, and pre-heat in the 65°C water bath.","checked":false,"checklist_id":957,"created_at":"2018-12-21T09:43:55.403Z","updated_at":"2018-12-21T09:43:55.403Z","created_by_id":null,"last_modified_by_id":null,"position":2},{"id":3937,"text":"Put the sample into the 65°C water bath and mix by inversion every 10 min for 30-60 minutes.","checked":false,"checklist_id":957,"created_at":"2018-12-21T09:43:55.412Z","updated_at":"2018-12-21T09:43:55.412Z","created_by_id":null,"last_modified_by_id":null,"position":3},{"id":3938,"text":"After incubation, centrifuge the sample tube for 5 min at 5000 × g and decant the supernatant into a new 50 mL Falcon tube.","checked":false,"checklist_id":957,"created_at":"2018-12-21T09:43:55.419Z","updated_at":"2018-12-21T09:43:55.419Z","created_by_id":null,"last_modified_by_id":null,"position":4}]}],"step_comments":[],"step_assets":[],"assets":[],"step_tables":[],"tables":[]},{"step":{"id":4417,"name":"Precipitation","description":"\u003c!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\"\u003e\n\u003chtml\u003e\u003cbody\u003e\u003cp\u003eIf you are precipitating small volumes of DNA, and you can fit the required amount of solvent into the sample tube, then ice-cold ethanol is the preferred choice. You can chill it (in liquid nitrogen or at \u003cstrong\u003e–80°C\u003c/strong\u003e) to accelerate the precipitation without the risk of precipitating excess salt. Afterwards, you need to wash the pellet with \u003cstrong\u003e70%\u003c/strong\u003e ethanol to remove any salt present.\u003c/p\u003e\u003c/body\u003e\u003c/html\u003e","position":2,"completed":false,"completed_on":null,"user_id":202,"created_at":"2018-11-12T12:05:42.004Z","updated_at":"2019-02-07T10:03:24.266Z","last_modified_by_id":202,"protocol_id":3487},"checklists":[{"checklist":{"id":959,"name":"Guidelines:","step_id":4417,"created_at":"2018-12-21T09:54:21.494Z","updated_at":"2018-12-21T09:54:21.494Z","created_by_id":null,"last_modified_by_id":null},"checklist_items":[{"id":3943,"text":"Add a ½ volume of 5 M NaCl to the sample and mix gently by inversion.","checked":false,"checklist_id":959,"created_at":"2018-12-21T09:54:21.496Z","updated_at":"2018-12-21T09:54:21.496Z","created_by_id":null,"last_modified_by_id":null,"position":0},{"id":3944,"text":"Then, add 3 volumes of cold 95% ethanol and mix gently by inversion.","checked":false,"checklist_id":959,"created_at":"2018-12-21T09:54:21.503Z","updated_at":"2018-12-21T09:54:21.503Z","created_by_id":null,"last_modified_by_id":null,"position":1},{"id":3945,"text":"Place the tubes into a -20°C freezer and incubate for 1 h.","checked":false,"checklist_id":959,"created_at":"2018-12-21T09:54:21.509Z","updated_at":"2018-12-21T09:54:21.509Z","created_by_id":null,"last_modified_by_id":null,"position":2},{"id":3946,"text":"After incubation, centrifuge the Falcon tube for 10 min at 5000 × g to pellet the DNA.","checked":false,"checklist_id":959,"created_at":"2018-12-21T09:54:21.516Z","updated_at":"2018-12-21T09:54:21.516Z","created_by_id":null,"last_modified_by_id":null,"position":3},{"id":3947,"text":"Carefully decant away the supernatant and wash the DNA pellet with 3 mL of 70% ethanol.","checked":false,"checklist_id":959,"created_at":"2018-12-21T09:54:21.522Z","updated_at":"2018-12-21T09:54:21.522Z","created_by_id":null,"last_modified_by_id":null,"position":4},{"id":3948,"text":"Gently swirl the solution and centrifuge again for 10 min at 5000 × g.","checked":false,"checklist_id":959,"created_at":"2018-12-21T09:54:21.529Z","updated_at":"2018-12-21T09:54:21.529Z","created_by_id":null,"last_modified_by_id":null,"position":5},{"id":3949,"text":"Carefully decant the supernatant and air-dry DNA pellet for 15 min at room temperature. Once dried, suspend DNA in 200 μL of TE buffer.","checked":false,"checklist_id":959,"created_at":"2018-12-21T09:54:21.535Z","updated_at":"2018-12-21T09:54:21.535Z","created_by_id":null,"last_modified_by_id":null,"position":6}]}],"step_comments":[],"step_assets":[],"assets":[],"step_tables":[],"tables":[]}]}],"results":[]}],"my_module_groups":[{"id":1167,"created_at":"2018-12-17T07:51:25.142Z","updated_at":"2018-12-17T07:51:25.142Z","created_by_id":202,"experiment_id":433}]} |