scinote-web/app/assets/templates/experiment_436/experiment.json

{"experiment":{"id":436,"name":"Transformation of E. coli with electroporation","description":"This template will show you how to transform E.coli bacteria with electroporation. In molecular biology,  transformation can be artificially induced through the creation of pores in the bacterial cell walls. \r\nElectroporation is the use of high-voltage electric shocks to introduce DNA into cells. It can be used with most cell types, yields a high frequency of both stable transformation and transient gene expression and, because it requires fewer steps, can be easier than alternate techniques.","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":"2018-11-13T09:57:55.843Z","updated_at":"2019-02-07T14:41:26.829Z","workflowimg_file_name":"wimg20181213-1-tptre.png","workflowimg_content_type":"image/png","workflowimg_file_size":3029,"workflowimg_updated_at":"2019-02-07T14:41:26.829Z","uuid":"59b17955-fba6-405a-a6b8-6a78a05af66f"},"my_modules":[{"my_module":{"id":2166,"name":"Analysis of transformation","due_date":null,"description":null,"x":67,"y":7,"my_module_group_id":1165,"created_at":"2018-11-13T15:51:41.812Z","updated_at":"2018-12-13T15:38:36.161Z","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":436,"state":"uncompleted","completed_on":null},"outputs":[],"my_module_tags":[],"task_comments":[],"my_module_repository_rows":[],"user_my_modules":[],"protocols":[{"protocol":{"id":3508,"name":null,"authors":null,"description":null,"added_by_id":null,"my_module_id":2166,"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":"2018-11-13T15:51:41.820Z","updated_at":"2019-02-20T07:58:02.380Z","published_on":null,"nr_of_linked_children":0},"protocol_protocol_keywords":[],"steps":[{"step":{"id":5189,"name":"Obtaining single colonies","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\u003eFor each electroporation, \u003cstrong\u003e2.5, 25\u003c/strong\u003e and \u003cstrong\u003e250 pl\u003c/strong\u003e of cells, each in duplicate, were spread on LB plates containing \u003cstrong\u003e12.5 mg/mL\u003c/strong\u003e chloramphenicol. For transformations of ligations with the vector, plates contained \u003cstrong\u003e50 µg/mL\u003c/strong\u003e X-gal and \u003cstrong\u003e25 µg/mL\u003c/strong\u003e IPTG.\u003cbr\u003e\u003cbr\u003e\u003cbr\u003e\u003cstrong\u003eGrowth conditions:\u003c/strong\u003e\u003c/p\u003e\n\u003cul\u003e\n\u003cli\u003e37°C\u003c/li\u003e\n\u003cli\u003e24 hours\u003c/li\u003e\n\u003c/ul\u003e\n\u003cbr\u003eScoring of colonies.\u003c/body\u003e\u003c/html\u003e","position":0,"completed":false,"completed_on":null,"user_id":202,"created_at":"2018-12-13T15:04:47.190Z","updated_at":"2019-02-19T14:21:06.329Z","last_modified_by_id":202,"protocol_id":3508},"checklists":[],"step_comments":[],"step_assets":[],"assets":[],"step_tables":[],"tables":[]},{"step":{"id":5190,"name":"Analysis","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\u003eThere are many factors that can influence transformation efficiency. Therefore after each transformation, an assessment of efficiency is needed.\u003cbr\u003e\u003cbr\u003e\u003cstrong\u003eFor gene expression common methods used: \u003c/strong\u003e\u003c/p\u003e\n\u003col\u003e\n\u003cli\u003eFlow Cytometry \u003c/li\u003e\n\u003cli\u003eqPCR\u003c/li\u003e\n\u003c/ol\u003e\n\u003cstrong\u003eFor protein expression common methods are:\u003cbr\u003e\u003c/strong\u003e\u003cbr\u003e\n\u003col\u003e\n\u003cli\u003eWestern blot analysis (\u003ca href=\"https://www.protocols.io/view/12-sds-page-western-blot-rwrd7d6\" target=\"_blank\" rel=\"noopener\"\u003eClick here\u003c/a\u003e)\u003c/li\u003e\n\u003cli\u003eMicroscopy (visualization of cells with your gene of interest)\u003c/li\u003e\n\u003c/ol\u003e\n\u003c/body\u003e\u003c/html\u003e","position":1,"completed":false,"completed_on":null,"user_id":202,"created_at":"2018-12-13T15:24:58.739Z","updated_at":"2019-02-20T07:58:02.245Z","last_modified_by_id":202,"protocol_id":3508},"checklists":[],"step_comments":[],"step_assets":[],"assets":[],"step_tables":[],"tables":[]}]}],"results":[]},{"my_module":{"id":2162,"name":"Storage of bacteria","due_date":null,"description":null,"x":0,"y":0,"my_module_group_id":1165,"created_at":"2018-11-13T10:11:52.799Z","updated_at":"2018-12-13T15:38:36.163Z","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":436,"state":"uncompleted","completed_on":null},"outputs":[{"id":5916,"input_id":2165,"output_id":2162}],"my_module_tags":[],"task_comments":[],"my_module_repository_rows":[],"user_my_modules":[],"protocols":[{"protocol":{"id":3504,"name":null,"authors":null,"description":null,"added_by_id":null,"my_module_id":2162,"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":"2018-11-13T10:11:52.850Z","updated_at":"2019-02-19T14:18:55.343Z","published_on":null,"nr_of_linked_children":0},"protocol_protocol_keywords":[],"steps":[{"step":{"id":4435,"name":"Collection and storage","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\u003eCulture samples are aliquoted to microfuge tubes (\u003cstrong\u003e100-200 µL/tube\u003c/strong\u003e) and frozen quickly in a dry ice-ethanol bath. Store at \u003cstrong\u003e-70°C\u003c/strong\u003e until use.\u003c/p\u003e\u003c/body\u003e\u003c/html\u003e","position":3,"completed":false,"completed_on":null,"user_id":202,"created_at":"2018-11-13T10:35:19.862Z","updated_at":"2019-02-07T12:58:44.529Z","last_modified_by_id":202,"protocol_id":3504},"checklists":[],"step_comments":[],"step_assets":[],"assets":[],"step_tables":[],"tables":[]},{"step":{"id":4438,"name":"Centrifugation","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\u003eFollow the steps for centrifugation below.\u003c/p\u003e\u003c/body\u003e\u003c/html\u003e","position":2,"completed":false,"completed_on":null,"user_id":202,"created_at":"2018-11-13T10:55:00.370Z","updated_at":"2018-12-24T08:25:52.273Z","last_modified_by_id":202,"protocol_id":3504},"checklists":[{"checklist":{"id":954,"name":"Guidelines:","step_id":4438,"created_at":"2018-12-21T07:49:43.577Z","updated_at":"2018-12-21T07:49:43.577Z","created_by_id":null,"last_modified_by_id":null},"checklist_items":[{"id":3917,"text":"Centrifuge samples for 10 min at 5000 r.p.m.","checked":false,"checklist_id":954,"created_at":"2018-12-21T07:49:43.580Z","updated_at":"2018-12-21T07:49:43.580Z","created_by_id":null,"last_modified_by_id":null,"position":0},{"id":3918,"text":"Resuspension in a volume of cold 10% sterile glycerol equal to the original culture volume.","checked":false,"checklist_id":954,"created_at":"2018-12-21T07:49:43.588Z","updated_at":"2018-12-21T07:49:43.588Z","created_by_id":null,"last_modified_by_id":null,"position":1},{"id":3919,"text":"Cells are collected by spinning for 10 min at 5000 r.p.m. at 4°C.","checked":false,"checklist_id":954,"created_at":"2018-12-21T07:49:43.595Z","updated_at":"2018-12-21T07:49:43.595Z","created_by_id":null,"last_modified_by_id":null,"position":2},{"id":3920,"text":"After decanting the supernatant, cells are resuspended in the volume of glycerol remaining in the centrifiuge bottles and spun for 10 min at 7000 r.p.m. in a centrifuge.","checked":false,"checklist_id":954,"created_at":"2018-12-21T07:49:43.604Z","updated_at":"2018-12-21T07:49:43.604Z","created_by_id":null,"last_modified_by_id":null,"position":3},{"id":3921,"text":"After decanting the supernatant, cells are resuspended in 10% glycerol, using a volume of between 2 and 2.25 m/L initial culture.","checked":false,"checklist_id":954,"created_at":"2018-12-21T07:49:43.611Z","updated_at":"2018-12-21T07:49:43.611Z","created_by_id":null,"last_modified_by_id":null,"position":4}]}],"step_comments":[],"step_assets":[],"assets":[],"step_tables":[],"tables":[]},{"step":{"id":4437,"name":"Incubation","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\u003cul\u003e\n\u003cli\u003eGrown at 37°C \u003c/li\u003e\n\u003cli\u003eShaking at 200 r.p.m. to an OD550 of 0.7\u003c/li\u003e\n\u003c/ul\u003e\u003c/body\u003e\u003c/html\u003e","position":1,"completed":false,"completed_on":null,"user_id":202,"created_at":"2018-11-13T10:44:55.407Z","updated_at":"2019-02-19T14:18:55.293Z","last_modified_by_id":202,"protocol_id":3504},"checklists":[],"step_comments":[],"step_assets":[],"assets":[],"step_tables":[],"tables":[]},{"step":{"id":4434,"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\u003eA fresh overnight culture of \u003cspan class=\"TextRun SCXO206656968\" lang=\"EN-US\" xml:lang=\"EN-US\"\u003e\u003cspan class=\"NormalTextRun SCXO206656968\"\u003e\u003cem\u003eEscherichia coli\u003c/em\u003e \u003c/span\u003e\u003c/span\u003eis diluted \u003cstrong\u003e1:1000\u003c/strong\u003e for growth in SOB medium. \u003cbr\u003eIncubation until density is \u003cstrong\u003e10\u003csup\u003e8 \u003c/sup\u003e- 2x10\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e8\u003c/strong\u003e \u003c/sup\u003e.\u003cbr\u003e\u003cbr\u003e\u003cbr\u003e\u003cstrong\u003eSOB medium:\u003c/strong\u003e\u003c/p\u003e\n\u003cul\u003e\n\u003cli\u003e2% (w/v) Bacto Tryptone\u003c/li\u003e\n\u003cli\u003e0,5% (w/v) yeast extract\u003c/li\u003e\n\u003cli\u003e10mM NaCl\u003c/li\u003e\n\u003cli\u003e2,5 mM KCl\u003c/li\u003e\n\u003cli\u003e10 mM MgCl2\u003c/li\u003e\n\u003cli\u003e10 mM MgS04\u003c/li\u003e\n\u003cli\u003eAutoclaved\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":"2018-11-13T10:23:13.538Z","updated_at":"2019-02-19T14:18:20.039Z","last_modified_by_id":202,"protocol_id":3504},"checklists":[],"step_comments":[],"step_assets":[],"assets":[],"step_tables":[],"tables":[]}]}],"results":[]},{"my_module":{"id":2165,"name":"Electroporation","due_date":null,"description":null,"x":33,"y":7,"my_module_group_id":1165,"created_at":"2018-11-13T12:54:50.584Z","updated_at":"2018-12-13T15:38:36.166Z","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":436,"state":"uncompleted","completed_on":null},"outputs":[{"id":5917,"input_id":2166,"output_id":2165}],"my_module_tags":[],"task_comments":[],"my_module_repository_rows":[],"user_my_modules":[],"protocols":[{"protocol":{"id":3507,"name":null,"authors":null,"description":null,"added_by_id":null,"my_module_id":2165,"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":"2018-11-13T12:54:50.592Z","updated_at":"2019-02-20T07:35:25.312Z","published_on":null,"nr_of_linked_children":0},"protocol_protocol_keywords":[],"steps":[{"step":{"id":4444,"name":"Preparation of material","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\u003eMark the required number of microcentrifuge tubes. Place the required number of cuvettes (\u003cstrong\u003e0.1 cm\u003c/strong\u003e gap) on ice. Before use remove the bacteria and aliquots of DNA from the freezer, thaw briefly at room temperature, then place on ice before electroporation. A mixture of cells and DNA: \u003cstrong\u003e30µL\u003c/strong\u003e of cells and\u003cstrong\u003e 2 µL\u003c/strong\u003e DNA is added to microfuge tubes.\u003cbr\u003e\u003cbr\u003e\u003c/p\u003e\u003c/body\u003e\u003c/html\u003e","position":0,"completed":false,"completed_on":null,"user_id":202,"created_at":"2018-11-13T12:56:40.301Z","updated_at":"2019-02-07T09:34:28.051Z","last_modified_by_id":202,"protocol_id":3507},"checklists":[],"step_comments":[],"step_assets":[],"assets":[],"step_tables":[],"tables":[]},{"step":{"id":4449,"name":"After electroporation","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\u003cdiv style=\"text-align: justify;\"\u003eInoculate samples in cuvettes with \u003cstrong\u003e0.5 mL\u003c/strong\u003e SOC is immediately after electroporation. \u003c/div\u003e\n\u003cdiv style=\"text-align: justify;\"\u003eContent is transferred to sterile glass culture tubes.\u003c/div\u003e\n\u003cul\u003e\n\u003cli\u003e\n\u003cdiv style=\"text-align: justify;\"\u003e45 minutes\u003c/div\u003e\n\u003c/li\u003e\n\u003cli\u003eShaking\u003c/li\u003e\n\u003cli\u003e37°C \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":"2018-11-13T15:48:41.251Z","updated_at":"2019-02-19T14:20:45.852Z","last_modified_by_id":202,"protocol_id":3507},"checklists":[],"step_comments":[],"step_assets":[],"assets":[],"step_tables":[],"tables":[]},{"step":{"id":4448,"name":"Electroporation","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\u003eUsing a micropipette, pipette \u003cstrong\u003e20 µL\u003c/strong\u003e of the cell-DNA mixture in cuvettes. Do not leave an air bubble in the droplet of cells; the pressure of a bubble may cause arcing and loss of the sample. Place the chamber in a slot and note its position. \u003cbr\u003eRepeat the process if more than one sample is to be pulsed. Handle the cuvettes gently to avoid accidentally displacing the sample. Close the lid and secure it with the draw latch. Set the electroporation settings.\u003cspan style=\"background-color: #f6d5d9;\"\u003e\u003cbr\u003e\u003c/span\u003e\u003cbr\u003eElectroporations are performed in duplicate.\u003cbr\u003e\u003cbr\u003e\u003cstrong\u003eConditions:\u003c/strong\u003e\u003c/p\u003e\n\u003cul\u003e\n\u003cli style=\"text-align: justify;\"\u003e100Ω\u003c/li\u003e\n\u003cli style=\"text-align: justify;\"\u003e1.8 kV\u003c/li\u003e\n\u003cli style=\"text-align: justify;\"\u003e25µF\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":"2018-11-13T15:19:26.546Z","updated_at":"2019-02-20T07:36:24.474Z","last_modified_by_id":202,"protocol_id":3507},"checklists":[],"step_comments":[],"step_assets":[{"id":3082,"step_id":4448,"asset_id":3744}],"assets":[{"id":3744,"created_at":"2019-02-20T07:35:25.074Z","updated_at":"2019-02-20T07:36:24.466Z","file_file_name":"Electroporation.jpg","file_content_type":"image/jpeg","file_file_size":42049,"file_updated_at":"2019-02-20T07:36:24.012Z","created_by_id":202,"last_modified_by_id":null,"estimated_size":46253,"file_present":true,"lock":null,"lock_ttl":null,"version":1,"file_processing":false,"team_id":1}],"step_tables":[],"tables":[]}]}],"results":[]},{"my_module":{"id":2163,"name":"Preparation of DNA","due_date":null,"description":null,"x":0,"y":16,"my_module_group_id":1165,"created_at":"2018-11-13T11:54:32.038Z","updated_at":"2018-12-13T15:38:36.168Z","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":436,"state":"uncompleted","completed_on":null},"outputs":[{"id":5918,"input_id":2165,"output_id":2163}],"my_module_tags":[],"task_comments":[],"my_module_repository_rows":[],"user_my_modules":[],"protocols":[{"protocol":{"id":3505,"name":null,"authors":null,"description":null,"added_by_id":null,"my_module_id":2163,"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":"2018-11-13T11:54:32.112Z","updated_at":"2019-02-19T14:17:49.489Z","published_on":null,"nr_of_linked_children":0},"protocol_protocol_keywords":[],"steps":[{"step":{"id":4441,"name":"Resuspension and storage","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\u003eThe plasmids were resuspended in \u003cstrong\u003e20µL\u003c/strong\u003e TE (\u003cstrong\u003e10 mM\u003c/strong\u003e tris-CI \u003cstrong\u003epH 8.0\u003c/strong\u003e, \u003cstrong\u003e1 mM EDTA\u003c/strong\u003e) and store in aliquots at \u003cstrong\u003e-20°C\u003c/strong\u003e.  \u003cbr\u003eDNA concentration is measured in two ways:\u003c/p\u003e\n\u003col\u003e\n\u003cli\u003eMeasuring \u003cstrong\u003eABS\u003csub\u003e260\u003c/sub\u003e\u003c/strong\u003e and calculating the concentration assuming a molar extinction coefficient of \u003cstrong\u003e1.3 x 10\u003csup\u003e4\u003c/sup\u003e L mol\u003csup\u003e-1\u003c/sup\u003e cm\u003csup\u003e-1\u003c/sup\u003e\u003c/strong\u003e (\u003cstrong\u003e1 ABS\u003csub\u003e260\u003c/sub\u003e unit = 50 µg/mL\u003c/strong\u003e). \u003c/li\u003e\n\u003cli\u003eLinearizing the plasmid DNA by restriction digestion and comparison of band intensity on an ethidium bromide-stained agarose gel with that of several other linear DNA species of known mass.\u003c/li\u003e\n\u003c/ol\u003e\n\u003c/body\u003e\u003c/html\u003e","position":2,"completed":false,"completed_on":null,"user_id":202,"created_at":"2018-11-13T12:30:26.870Z","updated_at":"2019-02-07T09:32:54.981Z","last_modified_by_id":202,"protocol_id":3505},"checklists":[],"step_comments":[],"step_assets":[],"assets":[],"step_tables":[],"tables":[]},{"step":{"id":5186,"name":"Growth of clones","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\u003ePlasmid DNA isolation procedure by Birnboim and Doly (1979). The method has been used principally with plasmid \u003cstrong\u003epBR322\u003c/strong\u003e and its derivatives in E.coli strains \u003cstrong\u003eHB101\u003c/strong\u003e, \u003cstrong\u003eRRI\u003c/strong\u003e and \u003cstrong\u003eSK 15921\u003c/strong\u003e. \u003cbr\u003e\u003cbr\u003e\u003c/p\u003e\u003c/body\u003e\u003c/html\u003e","position":0,"completed":false,"completed_on":null,"user_id":202,"created_at":"2018-12-13T09:33:57.218Z","updated_at":"2019-02-07T12:58:08.799Z","last_modified_by_id":202,"protocol_id":3505},"checklists":[{"checklist":{"id":955,"name":"Guidelines:","step_id":5186,"created_at":"2018-12-21T09:14:37.947Z","updated_at":"2018-12-21T09:14:37.947Z","created_by_id":null,"last_modified_by_id":null},"checklist_items":[{"id":3922,"text":"Selected clones are grown in 2.5 ml of L-broth containing 100 ug/ml ampicillin in 6-ml vials.","checked":false,"checklist_id":955,"created_at":"2018-12-21T09:14:37.950Z","updated_at":"2018-12-21T09:14:37.950Z","created_by_id":null,"last_modified_by_id":null,"position":0},{"id":3923,"text":"After 18 h incubation, 0.5 ml of culture is transferred to a 1.5 ml Eppendorf tube for plasmid extraction.","checked":false,"checklist_id":955,"created_at":"2018-12-21T09:14:37.957Z","updated_at":"2018-12-21T09:14:37.957Z","created_by_id":null,"last_modified_by_id":null,"position":1},{"id":3924,"text":"The remainder is stored at -20°C after the addition of glycerol to 40%.","checked":false,"checklist_id":955,"created_at":"2018-12-21T09:14:37.964Z","updated_at":"2018-12-21T09:14:37.964Z","created_by_id":null,"last_modified_by_id":null,"position":2}]}],"step_comments":[],"step_assets":[],"assets":[],"step_tables":[],"tables":[]},{"step":{"id":5187,"name":"Plasmid DNA isolation","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\u003eAll manipulations are carried out at room temperature unless otherwise indicated. \u003cbr\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cbr\u003e\u003cstrong\u003eLysozyme solution:\u003c/strong\u003e\u003cbr\u003e\u003cbr\u003e\n\u003cul\u003e\n\u003cli\u003ePrepare fresh daily from crystalline lysozyme and stock solutions of the other components.\u003c/li\u003e\n\u003cli\u003eStore at 0°C.\u003c/li\u003e\n\u003cli\u003e2 mg/ml lysozyme.\u003c/li\u003e\n\u003cli\u003e50 mM glucose.\u003c/li\u003e\n\u003cli\u003e10 mM CDTA.\u003c/li\u003e\n\u003cli\u003e25 mM Tris-HC1 (pH 8.0). \u003c/li\u003e\n\u003c/ul\u003e\n\u003cbr\u003e\u003cbr\u003e\u003cstrong\u003eAlkaline SDS solution:\u003c/strong\u003e\u003cbr\u003e\n\u003cul\u003e\n\u003cli\u003eStore at room temperature\u003c/li\u003e\n\u003cli\u003eStable for about 1 week\u003c/li\u003e\n\u003cli\u003e0.2 N NaOH\u003c/li\u003e\n\u003cli\u003e1% sodium dodecyl sulfate (SDS)\u003c/li\u003e\n\u003c/ul\u003e\n \u003cbr\u003e\u003cstrong\u003eHigh salt solution:\u003c/strong\u003e\u003cbr\u003e\u003cbr\u003e\n\u003cul\u003e\n\u003cli\u003eStore at room temperature.\u003c/li\u003e\n\u003cli\u003e3 M sodium acetate (pH 4.8).\u003c/li\u003e\n\u003cli\u003eAdjusting to pH 4.8 with glacial acetic acid.\u003c/li\u003e\n\u003cli\u003eAdjusting volume to 1 L.\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":"2018-12-13T10:48:53.179Z","updated_at":"2019-02-19T14:17:49.435Z","last_modified_by_id":202,"protocol_id":3505},"checklists":[{"checklist":{"id":956,"name":"Guidelines:","step_id":5187,"created_at":"2018-12-21T09:22:56.932Z","updated_at":"2018-12-21T09:22:56.932Z","created_by_id":null,"last_modified_by_id":null},"checklist_items":[{"id":3925,"text":"The tube is centrifuged for 15 seconds. The supernatant is carefully removed with a fine-tip aspirator and the cell pellet is thoroughly suspended in 100 ul of lysozyme solution.","checked":false,"checklist_id":956,"created_at":"2018-12-21T09:22:56.934Z","updated_at":"2018-12-21T09:22:56.934Z","created_by_id":null,"last_modified_by_id":null,"position":0},{"id":3926,"text":"After a 30 minute period of incubation at 0°C, 200 ml of alkaline SDS solution is added and the tube is gently vortexed. The suspension should become almost clear and slightly viscous.","checked":false,"checklist_id":956,"created_at":"2018-12-21T09:22:56.942Z","updated_at":"2018-12-21T09:22:56.942Z","created_by_id":null,"last_modified_by_id":null,"position":1},{"id":3927,"text":"The tube is maintained for 5 min at 0°C and then 150 ul of high-salt solution added. The contents of the tube are gently mixed by inversion for a few seconds during which time a clot of DNA forms.","checked":false,"checklist_id":956,"created_at":"2018-12-21T09:22:56.949Z","updated_at":"2018-12-21T09:22:56.949Z","created_by_id":null,"last_modified_by_id":null,"position":2},{"id":3928,"text":"The tube is maintained at 0°C for 60 min to allow most of the protein, high molecular weight RNA and chromosomal DNA to precipitate.","checked":false,"checklist_id":956,"created_at":"2018-12-21T09:22:56.957Z","updated_at":"2018-12-21T09:22:56.957Z","created_by_id":null,"last_modified_by_id":null,"position":3},{"id":3929,"text":"Centrifugation for 5 min yields an almost clear supernatant. Four-tenths of a ml of the supernatant is removed and transferred to a second centrifuge tube. Small amounts of floating material may be carried over at this time.","checked":false,"checklist_id":956,"created_at":"2018-12-21T09:22:56.964Z","updated_at":"2018-12-21T09:22:56.964Z","created_by_id":null,"last_modified_by_id":null,"position":4},{"id":3930,"text":"One ml of cold ethanol is added and the tube is held at -20°C for 30 min.","checked":false,"checklist_id":956,"created_at":"2018-12-21T09:22:56.971Z","updated_at":"2018-12-21T09:22:56.971Z","created_by_id":null,"last_modified_by_id":null,"position":5},{"id":3931,"text":"The precipitate is collected by centrifugation for 2 min and the supernatant removed by aspiration.","checked":false,"checklist_id":956,"created_at":"2018-12-21T09:22:56.977Z","updated_at":"2018-12-21T09:22:56.977Z","created_by_id":null,"last_modified_by_id":null,"position":6},{"id":3932,"text":"The pellet is dissolved in 100 ul of 0.1 M sodium acetate/0.05 M Tris-HCl (pH 8) and reprecipitated with 2 volumes of cold ethanol.","checked":false,"checklist_id":956,"created_at":"2018-12-21T09:22:56.984Z","updated_at":"2018-12-21T09:22:56.984Z","created_by_id":null,"last_modified_by_id":null,"position":7},{"id":3933,"text":"Redissolve once more in 100 ul of 0.1 M sodium acetate/0.05 M Tris-HCl (pH 8) and reprecipitated with 2 volumes of cold ethanol.","checked":false,"checklist_id":956,"created_at":"2018-12-21T09:22:56.992Z","updated_at":"2018-12-21T09:22:56.992Z","created_by_id":null,"last_modified_by_id":null,"position":8}]}],"step_comments":[],"step_assets":[],"assets":[],"step_tables":[],"tables":[]}]}],"results":[]}],"my_module_groups":[{"id":1165,"created_at":"2018-12-13T15:38:36.158Z","updated_at":"2018-12-13T15:38:36.158Z","created_by_id":202,"experiment_id":436}]}