scinote-web/app/assets/templates/experiment_422/experiment.json

{"experiment":{"id":422,"name":"CRISPR transformations with Agrobacterium tumefaciens","description":"This template will allow you to do CRISPR transformation of plants with Agrobacterium tumefaciens. The clustered regularly interspaced short palindromic repeats(CRISPR) associated Cas9/sgRNA system is a novel targeted genome-editing technique derived from the bacterial immune system. This technique enables precise genomic modifications in many different organisms and tissues.","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":"2018-11-08T10:28:00.553Z","updated_at":"2019-02-07T14:56:54.966Z","workflowimg_file_name":"wimg20181219-1-1wln20.png","workflowimg_content_type":"image/png","workflowimg_file_size":3309,"workflowimg_updated_at":"2019-02-07T14:56:54.966Z","uuid":"1a3d39cf-ea51-48a8-b622-d7a9492fd1e7"},"my_modules":[{"my_module":{"id":2089,"name":"Design and construction of gene-specific sgRNA","due_date":null,"description":null,"x":0,"y":0,"my_module_group_id":1182,"created_at":"2018-11-08T10:28:01.009Z","updated_at":"2018-12-19T11:33:27.356Z","archived":false,"archived_on":null,"created_by_id":202,"last_modified_by_id":null,"archived_by_id":null,"restored_by_id":null,"restored_on":null,"nr_of_assigned_samples":0,"workflow_order":0,"experiment_id":422,"state":"uncompleted","completed_on":null},"outputs":[{"id":5991,"input_id":2090,"output_id":2089}],"my_module_tags":[],"task_comments":[],"my_module_repository_rows":[],"user_my_modules":[],"protocols":[{"protocol":{"id":3391,"name":null,"authors":null,"description":null,"added_by_id":null,"my_module_id":2089,"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":"2018-11-08T10:28:01.077Z","updated_at":"2019-02-20T07:40:35.070Z","published_on":null,"nr_of_linked_children":0},"protocol_protocol_keywords":[],"steps":[{"step":{"id":4261,"name":"Use the online tools","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\u003ePaste selected sequence in the selected online tool. \u003cbr\u003e\u003cbr\u003eParameters that should be considered are:\u003cbr\u003e\n\u003c/p\u003e\n\u003cul\u003e\n\u003cli style=\"text-align: justify;\"\u003etarget length\u003c/li\u003e\n\u003cli style=\"text-align: justify;\"\u003ethe maximum number of tandemly repeated nucleotides\u003c/li\u003e\n\u003cli style=\"text-align: justify;\"\u003eminimum/maximum GC content. \u003c/li\u003e\n\u003c/ul\u003e\nSelect a genome to query for potential off-target recognition and analyze it. Evaluate and prioritize targets using sequence identity as well as off-target sequence identity.\u003c/body\u003e\u003c/html\u003e","position":1,"completed":false,"completed_on":null,"user_id":202,"created_at":"2018-11-08T10:28:01.386Z","updated_at":"2018-12-21T13:40:38.295Z","last_modified_by_id":202,"protocol_id":3391},"checklists":[],"step_comments":[],"step_assets":[],"assets":[],"step_tables":[],"tables":[]},{"step":{"id":4571,"name":"Considerations","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\u003eConsider choosing \u003cstrong\u003eseveral different target sites\u003c/strong\u003e, since not all sgRNAs work equally well. \u003cbr\u003eConsider using a \u003cstrong\u003e20 bp target site that also contains a recognition site of a commercially available restriction enzyme\u003c/strong\u003e. The target site of the restriction enzyme should span the site of Cas9 cleavage (\u003cstrong\u003e3 bp\u003c/strong\u003e in front of the PAM). This will allow you to use the restriction enzyme length polymorphism assay to detect mutations in your \u003cem\u003egene of interest.\u003c/em\u003e\u003c/p\u003e\u003c/body\u003e\u003c/html\u003e","position":2,"completed":false,"completed_on":null,"user_id":202,"created_at":"2018-11-15T13:25:17.662Z","updated_at":"2019-02-20T07:41:30.052Z","last_modified_by_id":202,"protocol_id":3391},"checklists":[],"step_comments":[],"step_assets":[{"id":3094,"step_id":4571,"asset_id":3756}],"assets":[{"id":3756,"created_at":"2019-02-20T07:40:34.833Z","updated_at":"2019-02-20T07:41:30.043Z","file_file_name":"Design_of_sgRNA.jpg","file_content_type":"image/jpeg","file_file_size":13667,"file_updated_at":"2019-02-20T07:41:29.670Z","created_by_id":202,"last_modified_by_id":null,"estimated_size":15033,"file_present":true,"lock":null,"lock_ttl":null,"version":1,"file_processing":false,"team_id":1}],"step_tables":[],"tables":[]},{"step":{"id":4570,"name":"Selecting genomic DNA region","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\u003eGenomic DNA region must correspond to the crRNA sequence of the sgRNA:\u003cbr\u003e\u003cbr\u003e\u003cstrong\u003eThe 3' end of the DNA target sequence must have a PAM sequence\u003c/strong\u003e. The \u003cstrong\u003e20 nucleotides\u003c/strong\u003e upstream of the PAM sequence will be your targeting sequence (crRNA), and Cas9 nuclease will cleave approximately three bases upstream of the PAM.\u003c/p\u003e\n\u003col\u003e\n\u003cli\u003eThe PAM sequence itself is absolutely required for cleavage, but it is NOT part of the sgRNA sequence and therefore should not be included in the sgRNA.\u003c/li\u003e\n\u003cli\u003eThe target sequence can be on either DNA strand.\u003c/li\u003e\n\u003c/ol\u003e\n\u003c/body\u003e\u003c/html\u003e","position":0,"completed":false,"completed_on":null,"user_id":202,"created_at":"2018-11-15T12:25:48.867Z","updated_at":"2019-02-20T07:40:29.591Z","last_modified_by_id":202,"protocol_id":3391},"checklists":[],"step_comments":[],"step_assets":[{"id":3093,"step_id":4570,"asset_id":3755}],"assets":[{"id":3755,"created_at":"2019-02-20T07:40:21.348Z","updated_at":"2019-02-20T07:40:29.583Z","file_file_name":"Genomic_target.jpg","file_content_type":"image/jpeg","file_file_size":14264,"file_updated_at":"2019-02-20T07:40:29.251Z","created_by_id":202,"last_modified_by_id":null,"estimated_size":15690,"file_present":true,"lock":null,"lock_ttl":null,"version":1,"file_processing":false,"team_id":1}],"step_tables":[],"tables":[]}]}],"results":[]},{"my_module":{"id":2090,"name":"Subcloning of the construct in plasmid","due_date":null,"description":null,"x":35,"y":0,"my_module_group_id":1182,"created_at":"2018-11-08T10:28:01.476Z","updated_at":"2018-12-19T11:33:27.359Z","archived":false,"archived_on":null,"created_by_id":202,"last_modified_by_id":null,"archived_by_id":null,"restored_by_id":null,"restored_on":null,"nr_of_assigned_samples":0,"workflow_order":1,"experiment_id":422,"state":"uncompleted","completed_on":null},"outputs":[{"id":5992,"input_id":2208,"output_id":2090}],"my_module_tags":[],"task_comments":[],"my_module_repository_rows":[],"user_my_modules":[],"protocols":[{"protocol":{"id":3393,"name":null,"authors":null,"description":null,"added_by_id":null,"my_module_id":2090,"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":"2018-11-08T10:28:01.537Z","updated_at":"2019-02-20T07:53:32.436Z","published_on":null,"nr_of_linked_children":0},"protocol_protocol_keywords":[],"steps":[{"step":{"id":4579,"name":"Verification","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\u003eVerify eight \u003cem\u003eE. coli\u003c/em\u003e colonies by colony PCR using primer and your protospacer primer with the following setup.\u003cbr\u003e\u003cbr\u003e\u003cstrong\u003ePCR program:\u003c/strong\u003e\u003c/p\u003e\n\u003cul\u003e\n\u003cli\u003e95°C 2 min\u003c/li\u003e\n\u003cli\u003e(35 cycles) 95°C 30 sec; 51°C 30 sec; 72°C 15 sec \u003c/li\u003e\n\u003cli\u003e72°C 15 sec\u003c/li\u003e\n\u003cli\u003e72°C 5 min\u003c/li\u003e\n\u003cli\u003e20°C \u003c/li\u003e\n\u003c/ul\u003e\n\u003cbr\u003e\u003cbr\u003eInoculate positive colony in LB medium supplemented with Amp and grow overnight.\u003c/body\u003e\u003c/html\u003e","position":3,"completed":false,"completed_on":null,"user_id":202,"created_at":"2018-11-15T14:54:15.366Z","updated_at":"2019-02-20T07:53:32.379Z","last_modified_by_id":202,"protocol_id":3393},"checklists":[],"step_comments":[],"step_assets":[],"assets":[],"step_tables":[{"id":461,"step_id":4579,"table_id":579}],"tables":[{"id":579,"created_at":"2018-11-15T14:54:15.369Z","updated_at":"2019-02-20T07:53:32.362Z","created_by_id":202,"last_modified_by_id":202,"name":"Mixture","team_id":1,"contents":"eyJkYXRhIjpbWyI1eCBSZWFjdGlvbiBidWZmZXIiLCI0IM68TCJdLFsicHJp\nbWVyICgxMM68TSkiLCIxzrxMIl0sWyJwcm90b3NwYWNlciBwcmltZXIgICgx\nMM68TSkiLCIxIM68TCJdLFsiZE5UUHMgKDEwbU0gZWFjaCkiLCIwLjTOvEwi\nXSxbImRkSDIwIiwiMTMuNM68TCJdLFsiVGFxICBwb2x5bWVyYXNlICg1IFUv\nzrxsKSIsIjAuMs68TCJdXX0=\n","data_vector":"JzAuMic6MzggJzAuNCc6MjUgJzEnOjExLDE5ICcxMCc6OCwxNiAnMTBtbSc6\nMjMgJzEzLjQnOjI5ICcyNzRsJzo2LDEzLDIxLDI3LDMxLDM3LDQwICcyNzRt\nJzoxMCwxOCAnMzE2Jzo1LDksMTIsMTcsMjAsMjYsMzAsMzYsMzkgJzQnOjQg\nJzUnOjM0ICc1eCc6MSAnYnVmZmVyJzozICdkZGgyMCc6MjggJ2RudHBzJzoy\nMiAnZWFjaCc6MjQgJ3BvbHltZXJhc2UnOjMzICdwcmltZXInOjcsMTUgJ3By\nb3Rvc3BhY2VyJzoxNCAncmVhY3Rpb24nOjIgJ3RhcSc6MzIgJ3UnOjM1\n"}]},{"step":{"id":4577,"name":"Transformation","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\u003eTransform competent \u003cem\u003eE. coli \u003c/em\u003ecells with the ligation reaction and spread the transformed cells on LB agar plates supplemented with Amp.\u003cbr\u003e\u003cbr\u003e\u003cbr\u003e\u003cstrong\u003eRecipe:\u003c/strong\u003e\u003cbr\u003e15 g/L Bacto-tryptone \u003cbr\u003e5 g/L yeast extract \u003cbr\u003e5 g/L NaCl \u003cbr\u003ewith 200 μg/mL ampicillin\u003c/p\u003e\u003c/body\u003e\u003c/html\u003e","position":2,"completed":false,"completed_on":null,"user_id":202,"created_at":"2018-11-15T14:38:34.247Z","updated_at":"2019-02-20T07:53:25.630Z","last_modified_by_id":202,"protocol_id":3393},"checklists":[],"step_comments":[],"step_assets":[],"assets":[],"step_tables":[],"tables":[]},{"step":{"id":4575,"name":"Digestion","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\u003eDigesting plasmid with a restriction enzyme for \u003cstrong\u003e3 hours at 37°C\u003c/strong\u003e. Run it on an agarose gel and extract the vector backbone from the gel using the DNA Purification Kit.\u003c/p\u003e\u003c/body\u003e\u003c/html\u003e","position":1,"completed":false,"completed_on":null,"user_id":202,"created_at":"2018-11-15T14:00:37.365Z","updated_at":"2019-02-07T13:01:07.241Z","last_modified_by_id":202,"protocol_id":3393},"checklists":[],"step_comments":[],"step_assets":[],"assets":[],"step_tables":[{"id":459,"step_id":4575,"table_id":577},{"id":540,"step_id":4575,"table_id":677}],"tables":[{"id":577,"created_at":"2018-11-15T14:16:48.703Z","updated_at":"2019-02-07T13:01:07.192Z","created_by_id":202,"last_modified_by_id":202,"name":"Restriction digest","team_id":1,"contents":"eyJkYXRhIjpbWyJwbGFzbWlkIiwiM868ZyJdLFsiMTB4IGJ1ZmZlciIsIjXO\nvEwiXSxbImRkSDIwIiwidG8gNDnOvEwiXSxbInJlc3RyaWN0aW9uIGVuenlt\nZSAoMjAgVS/OvGwpIiwiMc68TCJdXX0=\n","data_vector":"JzEnOjIxICcxMHgnOjUgJzIwJzoxNyAnMjc0Zyc6NCAnMjc0bCc6OSwxNCwy\nMCwyMyAnMyc6MiAnMzE2JzozLDgsMTMsMTksMjIgJzQ5JzoxMiAnNSc6NyAn\nYnVmZmVyJzo2ICdkZGgyMCc6MTAgJ2VuenltZSc6MTYgJ3BsYXNtaWQnOjEg\nJ3Jlc3RyaWN0aW9uJzoxNSAndG8nOjExICd1JzoxOA==\n"},{"id":677,"created_at":"2018-12-19T10:48:05.316Z","updated_at":"2019-02-07T13:01:07.218Z","created_by_id":202,"last_modified_by_id":202,"name":"Ligation","team_id":1,"contents":"eyJkYXRhIjpbWyJ2ZWN0b3IiLCIzMDAgbmciXSxbIkFubmVhbGVkIHByaW1l\ncnMiLCIxzrxMIl0sWyIxMHggbGlnYXRpb24gYnVmZmVyIiwiMs68TCJdLFsi\nZGRIMjAiLCJ0byAxOc68TCJdLFsiRE5BIGxpZ2FzZSIsIjHOvEwiXV19\n","data_vector":"JzEnOjYsMjIgJzEweCc6OSAnMTknOjE3ICcyJzoxMiAnMjc0bCc6OCwxNCwx\nOSwyNCAnMzAwJzoyICczMTYnOjcsMTMsMTgsMjMgJ2FubmVhbGVkJzo0ICdi\ndWZmZXInOjExICdkZGgyMCc6MTUgJ2RuYSc6MjAgJ2xpZ2FzZSc6MjEgJ2xp\nZ2F0aW9uJzoxMCAnbmcnOjMgJ3ByaW1lcnMnOjUgJ3RvJzoxNiAndmVjdG9y\nJzox\n"}]},{"step":{"id":4574,"name":"Generating double stranded DNA","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\u003eOrder forward and reverse primers for your candidate target sequence N\u003csub\u003e20\u003c/sub\u003e with overlaps for cloning and dilute them to a final concentration of \u003cstrong\u003e10 μM\u003c/strong\u003e.\u003cbr\u003eFor example:\u003cbr\u003eForward primer: TTCG-GN\u003csub\u003e19\u003c/sub\u003e\u003cbr\u003eReverse primer: AAAC-N19 \u003cbr\u003e\u003cbr\u003ePipette \u003cstrong\u003e10 μL\u003c/strong\u003e of each primer dilution into a \u003cstrong\u003e1.5 mL\u003c/strong\u003e microcentrifuge tube and mix.\u003cbr\u003e\u003cbr\u003e\u003cstrong\u003eHeat primer mix: \u003c/strong\u003e\u003c/p\u003e\n\u003cul\u003e\n\u003cli\u003e98 °C for 10 min\u003c/li\u003e\n\u003cli\u003e55 °C for 10 min\u003c/li\u003e\n\u003cli\u003eafterwards slowly cool down to room temperature  \u003c/li\u003e\n\u003c/ul\u003e\n\u003cbr\u003eBy doing so, a double-stranded DNA including your protospacer sequence will be generated.\u003c/body\u003e\u003c/html\u003e","position":0,"completed":false,"completed_on":null,"user_id":202,"created_at":"2018-11-15T13:57:43.072Z","updated_at":"2019-02-19T14:53:00.254Z","last_modified_by_id":202,"protocol_id":3393},"checklists":[],"step_comments":[],"step_assets":[],"assets":[],"step_tables":[],"tables":[]}]}],"results":[]},{"my_module":{"id":2208,"name":"Cloning of final plant vector","due_date":null,"description":null,"x":69,"y":0,"my_module_group_id":1182,"created_at":"2018-11-16T10:48:03.813Z","updated_at":"2018-12-19T11:33:27.361Z","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":422,"state":"uncompleted","completed_on":null},"outputs":[{"id":5993,"input_id":2091,"output_id":2208}],"my_module_tags":[],"task_comments":[],"my_module_repository_rows":[],"user_my_modules":[],"protocols":[{"protocol":{"id":3583,"name":null,"authors":null,"description":null,"added_by_id":null,"my_module_id":2208,"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":"2018-11-16T10:48:03.821Z","updated_at":"2019-02-19T14:55:34.883Z","published_on":null,"nr_of_linked_children":0},"protocol_protocol_keywords":[],"steps":[{"step":{"id":4621,"name":"Transformation of E.coli cells","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\u003eTransform competent \u003cem\u003eE. coli \u003c/em\u003ecells with the reaction mixture and spread the transformed cells on LB agar plates supplemented with Kan.\u003cbr\u003e\u003cstrong\u003e\u003cbr\u003eLB-medium:\u003c/strong\u003e\u003c/p\u003e\n\u003cul\u003e\n\u003cli\u003e15 g/L Bacto-tryptone\u003c/li\u003e\n\u003cli\u003e5 g/L yeast extract\u003c/li\u003e\n\u003cli\u003e5 g/L NaCl\u003c/li\u003e\n\u003cli\u003e30μg/mL kanamycin sulfate\u003c/li\u003e\n\u003c/ul\u003e\n\u003c/body\u003e\u003c/html\u003e","position":2,"completed":false,"completed_on":null,"user_id":202,"created_at":"2018-11-16T12:34:00.600Z","updated_at":"2019-02-19T14:54:57.484Z","last_modified_by_id":202,"protocol_id":3583},"checklists":[],"step_comments":[],"step_assets":[],"assets":[],"step_tables":[],"tables":[]},{"step":{"id":4618,"name":"Amplify the sgRNA cassette","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\u003eAmplify it from a plasmid with target sequence using primers and polymerase and then extract the sgRNA cassette from the gel. Assemble the plasmid backbone and the sgRNA cassette to generate the final vector using Assembly Master Mix according to the manufacturer’s instruction with an incubation time of \u003cstrong\u003e1 h\u003c/strong\u003e.\u003cbr\u003e\u003cbr\u003e\u003cstrong\u003ePCR program: \u003c/strong\u003e\u003cbr\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cul\u003e\n\u003cli\u003e98°C 30 seconds\u003c/li\u003e\n\u003cli\u003e35 cycles (98°C 15 sec; 69°C 30 sec; 72°C 20 sec)\u003c/li\u003e\n\u003cli\u003e72°C 5 min\u003c/li\u003e\n\u003cli\u003e20°C\u003c/li\u003e\n\u003c/ul\u003e\n\u003c/body\u003e\u003c/html\u003e","position":1,"completed":false,"completed_on":null,"user_id":202,"created_at":"2018-11-16T11:50:40.125Z","updated_at":"2019-02-19T14:54:39.448Z","last_modified_by_id":202,"protocol_id":3583},"checklists":[],"step_comments":[],"step_assets":[],"assets":[],"step_tables":[{"id":464,"step_id":4618,"table_id":583}],"tables":[{"id":583,"created_at":"2018-11-16T12:13:30.368Z","updated_at":"2019-02-19T14:54:39.422Z","created_by_id":202,"last_modified_by_id":202,"name":"Mixture","team_id":1,"contents":"eyJkYXRhIjpbWyI1eCBQaHVzaW9uIEhGLWJ1ZmZlciIsIjEwzrxMIl0sWyJG\nb3J3YXJkIHByaW1lciAoMTDOvE0pIiwiMi41zrxMIl0sWyJSZXZlcnNlIHBy\naW1lciAoMTDOvE0pIiwiMi41zrxMIl0sWyJkTlRQcyAoMTBtTSBlYWNoKSIs\nIjHOvEwiXSxbImRkSDJPIiwiMzPOvEwiXSxbInBGSDYgd2l0aCB0YXJnZXQg\nc2VxdWVuY2UgKDEwMCBuZy/OvGwpIiwiMC41zrxMIl0sWyJQaHVzaW9uIERO\nQSBwb2x5bWVyYXNlICgyVS/OvGwpIiwiMC41zrxMIl1dfQ==\n","data_vector":"JzAuNSc6NDMsNTIgJzEnOjI4ICcxMCc6NiwxMSwxOSAnMTAwJzozOSAnMTBt\nbSc6MjYgJzIuNSc6MTQsMjIgJzI3NGwnOjgsMTYsMjQsMzAsMzQsNDIsNDUs\nNTEsNTQgJzI3NG0nOjEzLDIxICcydSc6NDkgJzMxNic6NywxMiwxNSwyMCwy\nMywyOSwzMyw0MSw0NCw1MCw1MyAnMzMnOjMyICc1eCc6MSAnYnVmZmVyJzo1\nICdkZGgybyc6MzEgJ2RuYSc6NDcgJ2RudHBzJzoyNSAnZWFjaCc6MjcgJ2Zv\ncndhcmQnOjkgJ2hmJzo0ICdoZi1idWZmZXInOjMgJ25nJzo0MCAncGZoNic6\nMzUgJ3BodXNpb24nOjIsNDYgJ3BvbHltZXJhc2UnOjQ4ICdwcmltZXInOjEw\nLDE4ICdyZXZlcnNlJzoxNyAnc2VxdWVuY2UnOjM4ICd0YXJnZXQnOjM3ICd3\naXRoJzozNg==\n"}]},{"step":{"id":4623,"name":"Inoculation","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\u003eInoculate positive colony into LB medium supplemented with Kan and let grow overnight.\u003cbr\u003e\u003cbr\u003e\u003cstrong\u003eLB-medium:\u003cbr\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cul\u003e\n\u003cli\u003e15 g/L Bacto-tryptone\u003c/li\u003e\n\u003cli\u003e5 g/L yeast extract\u003c/li\u003e\n\u003cli\u003e5 g/L NaCl\u003c/li\u003e\n\u003cli\u003eWith 30μg/mL kanamycin sulfate\u003c/li\u003e\n\u003c/ul\u003e\n\u003c/body\u003e\u003c/html\u003e","position":4,"completed":false,"completed_on":null,"user_id":202,"created_at":"2018-11-16T12:53:26.057Z","updated_at":"2019-02-19T14:55:34.834Z","last_modified_by_id":202,"protocol_id":3583},"checklists":[],"step_comments":[],"step_assets":[],"assets":[],"step_tables":[],"tables":[]},{"step":{"id":4616,"name":"Digestion","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\u003eDigestion of a plasmid with restriction enzymes.\u003cbr\u003e\u003cbr\u003e\u003cstrong\u003eConditions:\u003c/strong\u003e\u003c/p\u003e\n\u003cul\u003e\n\u003cli\u003e3h\u003c/li\u003e\n\u003cli\u003e37°C\u003c/li\u003e\n\u003c/ul\u003e\n\u003cbr\u003eRun it on an agarose gel and extract the vector backbone from the gel using DNA Purification Kit.\u003c/body\u003e\u003c/html\u003e","position":0,"completed":false,"completed_on":null,"user_id":202,"created_at":"2018-11-16T10:52:10.517Z","updated_at":"2019-02-19T14:53:39.075Z","last_modified_by_id":202,"protocol_id":3583},"checklists":[],"step_comments":[],"step_assets":[],"assets":[],"step_tables":[{"id":463,"step_id":4616,"table_id":582}],"tables":[{"id":582,"created_at":"2018-11-16T10:52:10.520Z","updated_at":"2019-02-19T14:53:39.061Z","created_by_id":202,"last_modified_by_id":202,"name":"Digestion mixture","team_id":1,"contents":"eyJkYXRhIjpbWyJwbGFzbWlkIiwiM868ZyJdLFsiMTB4IGJ1ZmZlciIsIjXO\nvEwiXSxbImRkSDIwIiwidG8gNDjOvEwiXSxbInJlc3RyaWN0aW9uIGVuenlt\nZSAoMjAgVS/OvEwpIiwiMc68TCJdLFsicmVzdHJpY3Rpb24gZW56eW1lKDIw\nIFUvzrxMKSIsIjHOvEwiXV19\n","data_vector":"JzEnOjIxLDMwICcxMHgnOjUgJzIwJzoxNywyNiAnMjc0Zyc6NCAnMjc0bCc6\nOSwxNCwyMCwyMywyOSwzMiAnMyc6MiAnMzE2JzozLDgsMTMsMTksMjIsMjgs\nMzEgJzQ4JzoxMiAnNSc6NyAnYnVmZmVyJzo2ICdkZGgyMCc6MTAgJ2Vuenlt\nZSc6MTYsMjUgJ3BsYXNtaWQnOjEgJ3Jlc3RyaWN0aW9uJzoxNSwyNCAndG8n\nOjExICd1JzoxOCwyNw==\n"}]},{"step":{"id":4622,"name":"Verification","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\u003eVerify eight positive colonies by colony PCR.\u003cbr\u003e\u003cbr\u003e\u003cstrong\u003ePCR program:\u003c/strong\u003e\u003c/p\u003e\n\u003cul\u003e\n\u003cli\u003e95°C 2min\u003c/li\u003e\n\u003cli\u003e35 cycles (95°C 30sec; 52°C 30 sec; 72°C 45 sec)\u003c/li\u003e\n\u003cli\u003e72°C 5min\u003c/li\u003e\n\u003cli\u003e20°C\u003c/li\u003e\n\u003c/ul\u003e\n\u003c/body\u003e\u003c/html\u003e","position":3,"completed":false,"completed_on":null,"user_id":202,"created_at":"2018-11-16T12:46:27.019Z","updated_at":"2019-02-19T14:55:10.602Z","last_modified_by_id":202,"protocol_id":3583},"checklists":[],"step_comments":[],"step_assets":[],"assets":[],"step_tables":[{"id":465,"step_id":4622,"table_id":584}],"tables":[{"id":584,"created_at":"2018-11-16T12:46:27.024Z","updated_at":"2019-02-19T14:55:10.586Z","created_by_id":202,"last_modified_by_id":202,"name":"","team_id":1,"contents":"eyJkYXRhIjpbWyI1eCBSZWFjdGlvbiBCdWZmZXIiLCI0zrxMIl0sWyJGb3J3\nYXJkIHByaW1lciAoMTDOvE0pIiwiMc68TCJdLFsiUmV2ZXJzZSBwcmltZXIg\nKDEwzrxNKSIsIjHOvEwiXSxbImROVFBzICgxMG1NIGVhY2gpIiwiMCw0zrxM\nIl0sWyJkZEgyTyIsIjEzLDTOvEwiXSxbIlRhcSBHMiBwb2x5bWVyYXNlICg1\nVS/OvGwpIiwiMCwyzrxMIl0sWyJQaWNrIGhhbGYgb2YgYSBjb2xvbnkgd2l0\naCBwaXBldHRlIHRpcCBhbmQgc3VzcGVuZCBpdCBpbiB0aGUgUENSIHJlYWN0\naW9uIixudWxsXSxbIk90aGVyIGhhbGYgb2YgdGhlIGNvbG9ueSBpcyB1c2Vk\nIGZvciBpbm9jdWxhdGlvbiIsbnVsbF1dfQ==\n","data_vector":"JzAnOjI2LDQxICcxJzoxMiwyMCAnMTAnOjksMTcgJzEwbW0nOjI0ICcxMyc6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\nJzo1MA==\n"}]}]}],"results":[]},{"my_module":{"id":2091,"name":"Transformation of the plant","due_date":null,"description":null,"x":69,"y":18,"my_module_group_id":1182,"created_at":"2018-11-08T10:28:01.704Z","updated_at":"2018-12-19T11:33:27.364Z","archived":false,"archived_on":null,"created_by_id":202,"last_modified_by_id":null,"archived_by_id":null,"restored_by_id":null,"restored_on":null,"nr_of_assigned_samples":0,"workflow_order":3,"experiment_id":422,"state":"uncompleted","completed_on":null},"outputs":[{"id":5994,"input_id":2093,"output_id":2091}],"my_module_tags":[],"task_comments":[],"my_module_repository_rows":[],"user_my_modules":[],"protocols":[{"protocol":{"id":3395,"name":null,"authors":null,"description":null,"added_by_id":null,"my_module_id":2091,"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":"2018-11-08T10:28:01.760Z","updated_at":"2019-02-20T07:53:56.919Z","published_on":null,"nr_of_linked_children":0},"protocol_protocol_keywords":[],"steps":[{"step":{"id":4267,"name":"Transform competent A. tumefaciens cells","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\u003eWith \u003cstrong\u003e1 μg\u003c/strong\u003e of (pUB-Cas9-gene of interest) and spread the transformed cells on YEP agar plates supplemented with Rif/Gent/Kan.\u003cbr\u003e\u003cbr\u003e\u003cstrong\u003eRecipe:\u003c/strong\u003e \u003c/p\u003e\n\u003cul\u003e\n\u003cli\u003eYEP-medium\u003c/li\u003e\n\u003cli\u003e15 g/L yeast extract\u003c/li\u003e\n\u003cli\u003e10 g/L Bacto-peptone\u003c/li\u003e\n\u003cli\u003e5 g/L NaCl \u003c/li\u003e\n\u003c/ul\u003e\n\u003cbr\u003eWith \u003cstrong\u003e150 μg/mL\u003c/strong\u003e rifampicin, \u003cstrong\u003e50 μg/mL\u003c/strong\u003e gentamycin sulfate, \u003cstrong\u003e50 μg/mL\u003c/strong\u003e kanamycin.\u003cbr\u003e\u003cbr\u003e\u003cstrong\u003eCoculture growth:\u003c/strong\u003e\u003cbr\u003e\n\u003cul\u003e\n\u003cli\u003e2 days\u003c/li\u003e\n\u003cli\u003e30°C.\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":"2018-11-08T10:28:01.867Z","updated_at":"2019-02-19T14:56:03.660Z","last_modified_by_id":202,"protocol_id":3395},"checklists":[],"step_comments":[],"step_assets":[],"assets":[],"step_tables":[],"tables":[]},{"step":{"id":4626,"name":"Verification of plasmid presence","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\u003eVerify plasmid presence in at least three colonies by colony PCR.\u003c/p\u003e\n\u003cbr\u003e\u003cstrong\u003ePCR program:\u003c/strong\u003e\u003cbr\u003e\n\u003cul\u003e\n\u003cli\u003e95°C 2min \u003c/li\u003e\n\u003cli\u003e35 cycles (95°C 30 sec; 60°C 30 sec; 72°C 1 min)\u003c/li\u003e\n\u003cli\u003e72°C 5 min\u003c/li\u003e\n\u003cli\u003e20°C\u003c/li\u003e\n\u003c/ul\u003e\n\u003c/body\u003e\u003c/html\u003e","position":1,"completed":false,"completed_on":null,"user_id":202,"created_at":"2018-11-16T13:23:32.534Z","updated_at":"2019-02-20T07:53:56.784Z","last_modified_by_id":202,"protocol_id":3395},"checklists":[{"checklist":{"id":971,"name":"Guideline:","step_id":4626,"created_at":"2018-12-21T14:16:54.698Z","updated_at":"2018-12-21T14:16:54.698Z","created_by_id":null,"last_modified_by_id":null},"checklist_items":[{"id":3998,"text":"In order to verify colony, pick half of a colony with a pipette tip and suspend cells in a 1.5 mL microcentrifuge tube containing 100 μL ddH2O.","checked":false,"checklist_id":971,"created_at":"2018-12-21T14:16:54.700Z","updated_at":"2018-12-21T14:16:54.700Z","created_by_id":null,"last_modified_by_id":null,"position":0},{"id":3999,"text":"Heat to 98 °C for 10 min and use 2 μL as DNA template for the PCR.","checked":false,"checklist_id":971,"created_at":"2018-12-21T14:16:54.707Z","updated_at":"2018-12-21T14:16:54.707Z","created_by_id":null,"last_modified_by_id":null,"position":1},{"id":4000,"text":"Streak the other half of the colony on a fresh YEP agar plate supplemented with Rif/Gent/Kan.","checked":false,"checklist_id":971,"created_at":"2018-12-21T14:16:54.715Z","updated_at":"2018-12-21T14:16:54.715Z","created_by_id":null,"last_modified_by_id":null,"position":2},{"id":4001,"text":"Grow cells at 30 °C for one day and then store at 4 °C.","checked":false,"checklist_id":971,"created_at":"2018-12-21T14:16:54.721Z","updated_at":"2018-12-21T14:16:54.721Z","created_by_id":null,"last_modified_by_id":null,"position":3}]}],"step_comments":[],"step_assets":[],"assets":[],"step_tables":[{"id":466,"step_id":4626,"table_id":585}],"tables":[{"id":585,"created_at":"2018-11-16T14:34:51.901Z","updated_at":"2019-02-20T07:53:56.770Z","created_by_id":202,"last_modified_by_id":202,"name":"PCR mixture","team_id":1,"contents":"eyJkYXRhIjpbWyI1eCBSZWFjdGlvbiBidWZmZXIiLCI0zrxMIl0sWyJGb3J3\nYXJkIFByaW1lciAoMTDOvE0pIiwiMc68TCJdLFsiUmV2ZXJzZSBwcmltZXIg\nKDEwzrxNKSIsIjHOvEwiXSxbImROVFBzICgxMG1NIGVhY2gpIiwiMC40zrxM\nIl0sWyJkZEgyMCIsIjExLjTOvEwiXSxbIkROQSB0ZW1wbGF0ZSIsIjLOvEwi\nXSxbIlRhcSBwb2x5bWVyYXNlICg1VS/OvGwpIiwiMC4yzrxMIl1dfQ==\n","data_vector":"JzAuMic6NDMgJzAuNCc6MjYgJzEnOjEyLDIwICcxMCc6OSwxNyAnMTBtbSc6\nMjQgJzExLjQnOjMwICcyJzozNSAnMjc0bCc6NiwxNCwyMiwyOCwzMiwzNyw0\nMiw0NSAnMjc0bSc6MTEsMTkgJzMxNic6NSwxMCwxMywxOCwyMSwyNywzMSwz\nNiw0MSw0NCAnNCc6NCAnNXUnOjQwICc1eCc6MSAnYnVmZmVyJzozICdkZGgy\nMCc6MjkgJ2RuYSc6MzMgJ2RudHBzJzoyMyAnZWFjaCc6MjUgJ2ZvcndhcmQn\nOjcgJ3BvbHltZXJhc2UnOjM5ICdwcmltZXInOjgsMTYgJ3JlYWN0aW9uJzoy\nICdyZXZlcnNlJzoxNSAndGFxJzozOCAndGVtcGxhdGUnOjM0\n"}]},{"step":{"id":4627,"name":"Transformation of the plants","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\u003eTransform plants using \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated T-DNA transfer with \u003cstrong\u003ethe floral dipping method\u003c/strong\u003e.\u003cbr\u003eSimple dipping of developing floral tissues into a solution containing \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e, \u003cstrong\u003e5%\u003c/strong\u003e sucrose and \u003cstrong\u003e500\u003c/strong\u003e microliters per litre of surfactant Silwet L‐77. Sucrose and surfactant are critical to the success of the floral dip method. Plants inoculated when numerous immature floral buds and few siliques are present produce transformed progeny at the highest rate. \u003c/p\u003e\u003c/body\u003e\u003c/html\u003e","position":2,"completed":false,"completed_on":null,"user_id":202,"created_at":"2018-11-16T14:41:33.494Z","updated_at":"2019-02-20T07:41:30.530Z","last_modified_by_id":202,"protocol_id":3395},"checklists":[],"step_comments":[],"step_assets":[{"id":3095,"step_id":4627,"asset_id":3757}],"assets":[{"id":3757,"created_at":"2019-02-20T07:40:57.546Z","updated_at":"2019-02-20T07:41:30.518Z","file_file_name":"Floral_dipping_method.jpg","file_content_type":"image/jpeg","file_file_size":16842,"file_updated_at":"2019-02-20T07:41:30.083Z","created_by_id":202,"last_modified_by_id":null,"estimated_size":18526,"file_present":true,"lock":null,"lock_ttl":null,"version":1,"file_processing":false,"team_id":1}],"step_tables":[],"tables":[]}]}],"results":[]},{"my_module":{"id":2093,"name":"Screening and Selection","due_date":null,"description":null,"x":35,"y":18,"my_module_group_id":1182,"created_at":"2018-11-08T10:28:02.085Z","updated_at":"2018-12-19T11:33:27.366Z","archived":false,"archived_on":null,"created_by_id":202,"last_modified_by_id":null,"archived_by_id":null,"restored_by_id":null,"restored_on":null,"nr_of_assigned_samples":0,"workflow_order":4,"experiment_id":422,"state":"uncompleted","completed_on":null},"outputs":[],"my_module_tags":[],"task_comments":[],"my_module_repository_rows":[],"user_my_modules":[],"protocols":[{"protocol":{"id":3399,"name":null,"authors":null,"description":null,"added_by_id":null,"my_module_id":2093,"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":"2018-11-08T10:28:02.138Z","updated_at":"2019-02-20T07:41:16.226Z","published_on":null,"nr_of_linked_children":0},"protocol_protocol_keywords":[],"steps":[{"step":{"id":5234,"name":"Screening of transformed plants","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\u003ePCR/RE Genotyping: Genotyping of clones with targeted mutation\u003cbr\u003eDNA sequencing\u003c/p\u003e\u003c/body\u003e\u003c/html\u003e","position":0,"completed":false,"completed_on":null,"user_id":202,"created_at":"2018-12-19T11:28:44.151Z","updated_at":"2018-12-19T11:29:48.670Z","last_modified_by_id":202,"protocol_id":3399},"checklists":[],"step_comments":[],"step_assets":[],"assets":[],"step_tables":[],"tables":[]},{"step":{"id":4270,"name":"Selection of genetically edited plant with desired modification","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\u003eThe transgene free homozygous mutants with desired genetic modifications at the targeted loci could be selected by selfing of GE0 generation plants and after segregation of the transgene in the next GE1 generation. The GE plants could be selected by PCR/RE genotyping and DNA sequencing of clones and negatively selecting for the transgene free plants with desired modification in the first generation only.\u003c/p\u003e\u003c/body\u003e\u003c/html\u003e","position":1,"completed":false,"completed_on":null,"user_id":202,"created_at":"2018-11-08T10:28:02.155Z","updated_at":"2019-02-20T07:41:31.063Z","last_modified_by_id":202,"protocol_id":3399},"checklists":[],"step_comments":[],"step_assets":[{"id":3096,"step_id":4270,"asset_id":3758}],"assets":[{"id":3758,"created_at":"2019-02-20T07:41:15.986Z","updated_at":"2019-02-20T07:41:31.054Z","file_file_name":"Selection.jpg","file_content_type":"image/jpeg","file_file_size":32384,"file_updated_at":"2019-02-20T07:41:30.581Z","created_by_id":202,"last_modified_by_id":null,"estimated_size":35622,"file_present":true,"lock":null,"lock_ttl":null,"version":1,"file_processing":false,"team_id":1}],"step_tables":[],"tables":[]}]}],"results":[]}],"my_module_groups":[{"id":1182,"created_at":"2018-12-19T11:33:27.354Z","updated_at":"2018-12-19T11:33:27.354Z","created_by_id":202,"experiment_id":422}]}