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Protocol ID: 1022

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Protocol ID: 4096

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Protocol ID: 5038

{"api_version":"1","is_new_mode":"1","protocol_name":"Transformation of Skeletonema marinoi using Multipulse Electroporation","last_modified":"1487441541","type_id":"1","link":"","fork_id":"","public_fork_note":"","number_of_steps":"7","uri":"transformation-of-skeletonema-marinoi-using-multip-g6nbzde","has_versions":"0","first_published_date":"2017-02-10 10:06:56","publish_date":"2017-02-10 10:06:56","documents":null,"have_protocol_in_step":"0","is_protocol_in_step":"0","vendor_name":"Contributed by users","vendor_link":"https://www.protocols.io","vendor_logo":"/img/vendors/1.png","mod_mins":"-44","mod_secs":"55","description":"<p><em>The following transformation protocol is designed for the insertion of linear DNA constructs into the nuclear genome of Skeletonema marinoi by non-homologous recombination.</em></p>","is_bookmarked":"0","can_reassign":"0","before_start":"","has_guidelines":"0","materials":null,"warning":"","is_prepublished":"0","public":"1","is_owner":"0","is_original_owner":"0","created_on":"1486720407","authors":"Oskar N. Johansson., Adrian K. Clarke","protocol_affiliation":"BioEnv - University of Gothenburg","steps":[{"id":"319256","is_changed":"0","original_id":"0","guid":"3A674EEDAB454907BEAB82DCDE053595","previous_guid":null,"components":[{"component_type_id":"1","name":"Description","data":"<p>a) Grow two 400 mL cultures in Artificial Seawater with f/2+Si supplements (Growth media) for ca. one week until dense. The standard growth conditions used in our lab are 16°C, 50-70 µmol. photons m-2 s-1, 16 h photoperiod.</p>\n<p>b) Reduce the volume of the sedimented culture in each flask to 50 mL by suction. Resuspend cells by vigorous agitation to minimize chain length and then transfer them to a 50 mL centrifuge tube.</p>\n<p>c) Pellet cells by centrifugation (1200 x g, 5 min, 4°C, swing-out rotor). Decant supernatant and resuspend the pellet in each tube in 10 mL of ice-cold 0.3M sorbitol.</p>\n<p>d) Pellet cells once more by centrifugation (1200 x g, 5 min, 4°C, swing-out rotor) and resuspend in 2.5 mL of ice-cold 0.3M sorbitol. Pool both resuspensions in a single tube.</p>\n<p>e) Store the cell suspension on ice until needed. </p>","data_id":"0"},{"component_type_id":"6","name":"Section","data":"Cell preparation","data_id":"0"},{"component_type_id":"13","name":"Annotation","data":"","data_id":"14676","source_data":[{"annotation_id":"14676","id":"14676","uri":"annotation-on-step-1-of-transformation-of-skeletonema","step_id":"319256","protocol_uri":"transformation-of-skeletonema-marinoi-using-multip-g6nbzde","protocol_name":"Transformation of Skeletonema marinoi using Multipulse Electroporation","annotation":"<p>Test</p>","body":"<p>Test</p>","user_id":"23187","original_user_id":"14012","can_edit":"0","can_delete":"0","show_name":"1","created_date":"1487371029","created_on":"1487371029","last_updated":null,"profile_image":"/img/avatars/017.png","full_name":"Jon Udell","affiliation":null,"username":"jon-udell","email":"[email protected]","pa_id":"14012","pa_useranme":"adrian-clarke","comments":[]}]}]},{"id":"319300","is_changed":"0","original_id":"0","guid":"0B729134C6634465829EEAD65EEDBA48","previous_guid":"3A674EEDAB454907BEAB82DCDE053595","components":[{"component_type_id":"1","name":"Description","data":"<p><em>This protocol was tested using the Gene Pulser Xcell electroporator (BioRad, USA) with the recommended 2 mm cuvettes (BioRad, USA).</em></p>","data_id":"0"},{"component_type_id":"6","name":"Section","data":"ElectroporatEquipment","data_id":"0"}]},{"id":"319306","is_changed":"0","original_id":"0","guid":"4ED25CA215DA47A68682C9F63B759156","previous_guid":"0B729134C6634465829EEAD65EEDBA48","components":[{"component_type_id":"1","name":"Description","data":"<p>Add 3-5 μg of the linear DNA construct to a 100 μL cell suspension in a 2 mm electroporation cuvette and leave for 3-5 min at 4°C. <br /><br /><em>NB. DNA construct should include a selectable marker such as antibiotic resistance (e.g., zeocin/bleomycin)</em></p>","data_id":"0"},{"component_type_id":"6","name":"Section","data":"Combining the cell suspension with desired DNA","data_id":"0"}]},{"id":"319314","is_changed":"0","original_id":"0","guid":"1EB656469EB44888A9119D250D34DEE2","previous_guid":"4ED25CA215DA47A68682C9F63B759156","components":[{"component_type_id":"1","name":"Description","data":"<p>Ensure cells are resuspended in the cuvette prior to electroporation. Electroporation is performed by the following steps:</p>\n<p> </p>\n<p>a) Poring pulses (300 V, 6 pulses, 1 sec pulse Interval, 5 ms pulse length)</p>\n<p>b) Transfer pulses (40 pulses [10x4], 50 ms pulse length, 0.1 s pulse interval)</p>\n<p>c) Reverse direction of the cuvette. </p>\n<p>d) Repeat transfer pulses (40 pulses [10x4], 50 ms pulse length, 0.1 s pulse interval)</p>\n<p> </p>","data_id":"0"},{"component_type_id":"6","name":"Section","data":"Electroporation","data_id":"0"}]},{"id":"319387","is_changed":"0","original_id":"0","guid":"5DA068342E714C158A41C5433AC9EC64","previous_guid":"1EB656469EB44888A9119D250D34DEE2","components":[{"component_type_id":"1","name":"Description","data":"<p>Transfer the electroporated cells from the cuvette using a Pasteur pipette to 30 mL liquid growth media (without selection) and leave for 48 h under standard growth conditions</p>","data_id":"0"},{"component_type_id":"6","name":"Section","data":"Cell recovery","data_id":"0"}]},{"id":"319391","is_changed":"0","original_id":"0","guid":"4D3CD4C8434C4143A4DEFCD344132F8A","previous_guid":"5DA068342E714C158A41C5433AC9EC64","components":[{"component_type_id":"1","name":"Description","data":"<p>Add selection to the media and continue growth for another 48 h</p>","data_id":"0"},{"component_type_id":"6","name":"Section","data":"Apply Selection","data_id":"0"}]},{"id":"319398","is_changed":"0","original_id":"0","guid":"ECA2E8DAD3BC4EE698268C16F9FAB9C7","previous_guid":"4D3CD4C8434C4143A4DEFCD344132F8A","components":[{"component_type_id":"1","name":"Description","data":"<p>a) Pellet cells in 50 mL centrifuge tubes (1200 x g, 5 min, room temperature) and resuspend in 1 mL growth medium with selection.</p>\n<p>b) Spread 75 (for 7 cm diameter plates) or 250 µL (for 15 cm diameter plates) of the resuspension on growth media plates (f/2 + Si + selection, 0.9% Agar).</p>\n<p> </p>\n<p><em>Left under standard growth conditions, colonies should appear after 2-3 weeks. </em></p>","data_id":"0"},{"component_type_id":"6","name":"Section","data":"Transfer to Solid media","data_id":"0"}]}],"doi":"dx.doi.org/10.17504/protocols.io.g6nbzde","doi_status":"2","version_id":"0","changed_fork_steps":null,"profile_url":"w2x234w2r2","protocol_img":"https://s3.amazonaws.com/pr-journal/ia2dpr6.jpg","profile_image":"/img/avatars/002.png","full_name":"Adrian Clarke","private_link":"87879142B7D5A76958A2335598E3456A","original_img":"1","username":"adrian-clarke","is_retracted":"0","retraction_reason":null,"first_name":"Adrian","last_name":"Clarke","affiliation":"","created_by":"Adrian Clarke","groups":[{"group_id":"108","group_uri":"protist-research-to-optimize-tools-in-genetics-protg","group_name":"Protist Research to Optimize Tools in Genetics (PROT-G)","group_logo":"https://s3.amazonaws.com/pr-journal/e3addmn.png"}],"big_protocol_img":"https://s3.amazonaws.com/pr-journal/iazdpr6.jpg","big_protocol_img_ofn":"skel.jpg","number_of_comments":4,"number_of_shared_runs":0,"tags":[],"ownership_history":[{"change_time":"2017-02-10 11:52:22","username":"oskar-johansson","profile_image":"https://s3.amazonaws.com/pr-journal/icvdpr6.jpg","full_name":"Oskar Johansson","affiliation":"Biology and Environmental Sciences - Gothenburg University"}],"collections":[],"keywords":"","contact_badges":[],"banner":null}