PCR conditions:
\nLibraries can be quantified by fluorimetry and calibrated by sequencing.
", "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": [ { "asset": { "id": 3746, "created_at": "2019-02-20T07:36:46.773Z", "updated_at": "2019-02-20T07:37:25.383Z", "created_by_id": 202, "last_modified_by_id": null, "estimated_size": 19765, "lock": null, "lock_ttl": null, "version": 1, "file_processing": false, "team_id": 1 }, "asset_blob": { "filename": "Shearing.jpg" } }, { "asset": { "id": 3747, "created_at": "2019-02-20T07:36:46.946Z", "updated_at": "2019-02-20T07:37:25.757Z", "created_by_id": 202, "last_modified_by_id": null, "estimated_size": 16540, "lock": null, "lock_ttl": null, "version": 1, "file_processing": false, "team_id": 1 }, "asset_blob": { "filename": "Amplification.jpg" } } ], "step_tables": [], "tables": [] }, { "step": { "id": 4420, "name": "RAD library preparation", "description": "\n\n The RAD library was prepared as originally described in article.
At least 30 specimens need to be chosen.
Each sample is digested at 37°C for 40 min with restriction enzyme:
Reads of low quality must be discarded.
Retained 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.
Parameters:
\n
Sequencing 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/adegenet package for Principal Component Analysis (PCA) and Discriminant Analysis of Principal Components (DAPC). PCA creates simplified models of the total variation within the dataset. DAPC identifies clusters of genetically related individuals.
", "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, "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": "\nReading 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.
", "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": "\n\nSNP of interest at a given locus need to be at least 20 bp from the end of a given sequence, to allow for primer design. SNP assays were designed and manufactured for use with KASP genotyping technology.
Optimisation assay conditions:
Here are the guidelines for protein extraction:
", "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": "\n\nLooking for a single absorbance peak at 260 nm, a 260/280 absorbance ratio of 1.8-2.0 and no evidence of substantial band shearing or contamination (either RNA or polysaccharide).
Assess the quality of the extracted DNA:
\nGuidelines for samples preparation below.
", "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": "\nIf 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 –80°C) to accelerate the precipitation without the risk of precipitating excess salt. Afterwards, you need to wash the pellet with 70% ethanol to remove any salt present.
", "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 } ] }