def main(p, params): """This protocol takes a tube of bacteria, amplifies it, and creates/stores new tubes of frozen cells """ #bacterial protocol p.mammalian_cell_mode = False amplify_and_freeze_bacteria( p, params['bacteria_well'], Antibiotic.from_string(params['antibiotic']) if params['antibiotic'] != 'cell_line' else None, Antibiotic.from_string(params['second_antibiotic']) if params['second_antibiotic'] != 'cell_line' else None, params['destroy_source_tube'])
def main(p, params): """This protocol takes a tube of bacteria, amplifies it, pellets the results. It can optionally induce additional BAC plasmids with IPTG """ #bacterial protocol p.mammalian_cell_mode = False pellet_bacteria(p, params['bacteria_well'], Antibiotic.from_string(params['antibiotic']) if params['antibiotic'] != 'cell_line' else None, params['destroy_source_tube'], params['induce_high_copy_number'] )
def main(p, params): """This protocol takes a tube of bacteria, amplifies it, and creates/stores new tubes of frozen cells """ #bacterial protocol p.mammalian_cell_mode = False miniprep(p, params['bacteria_well'], antibiotic=Antibiotic.from_string(params['antibiotic']) if params['antibiotic'] != 'cell_line' else None, destroy_source_tube=params['destroy_source_tube'], incubate_hours=params['incubate_hours'], measure_od=params['measure_od'], induce_high_copy_number=params['induce_high_copy_number'], slow_growth=params['slow_growth'] )
def amplify_and_freeze_bacteria(p, source_bacteria_well, antibiotic=None, second_antibiotic=None, destroy_source_tube=False): cell_line_name = get_cell_line_name(source_bacteria_well) assert isinstance(p, Protocol) if not antibiotic: #get the antibiotic from the source well well_antibiotic_str = source_bacteria_well.properties.get('antibiotic') if well_antibiotic_str: antibiotic = Antibiotic.from_string(well_antibiotic_str) else: raise Exception( 'Source Well must have property \'antibiotic\' set if antibiotic not specified' ) assert isinstance(antibiotic, Antibiotic) assert antibiotic != second_antibiotic, "can't add the same antibiotic twice" # Tubes and plates growth_plate = p.ref('growth_plate', cont_type="96-flat", discard=True) growth_wells = growth_plate.wells(['A1', 'B1']) set_well_name(growth_wells, 'growth_well') control_well = growth_plate.wells(['C1']) control_well.name = 'blanking_well' mix_well = p.ref('temp_mix_tube', cont_type='micro-1.5', discard=True).well(0) p.transfer(source_bacteria_well, mix_well, ul(25), mix_before=True) #adding antibiotic mixes after by default p.add_antibiotic(mix_well, antibiotic, broth_volume=ul(650)) if second_antibiotic: p.add_antibiotic(mix_well, second_antibiotic) p.distribute(mix_well, growth_wells, ul(325), allow_carryover=True) p.cover(growth_plate) #grow bacteria until they are in their log phase of growth #https://www.qiagen.com/us/resources/technologies/plasmid-resource-center/growth%20of%20bacterial%20cultures/ p.incubate(growth_plate, "warm_37", "{}:hour".format(16), shaking=True) p.add_antibiotic(control_well, antibiotic, broth_volume=ul(325), mix_after=False) p.measure_bacterial_density(growth_wells + control_well, one_tip=True, mix_before=True) mix_well2 = p.ref('temp_mix_tube2', cont_type='micro-1.5', discard=True).well(0) #add glycerol p.provision_by_name(Reagent.glycerol, mix_well2, ul(600)) p.consolidate(growth_wells, mix_well2, get_volume(growth_wells[0], aspiratable=True), mix_after=True) cryo_vial_wells = [] for x in range(0, 10): cryo_vial_wells.append( p.ref("cryo_frozen_%s_cells_%s" % (cell_line_name, x), cont_type="micro-1.5", storage="cold_80", cell_line_name=cell_line_name).well(0)) copy_cell_line_name(source_bacteria_well, cryo_vial_wells) set_property(cryo_vial_wells, 'antibiotic', antibiotic.name) p.distribute(mix_well2, cryo_vial_wells, ul(115), allow_carryover=True) if destroy_source_tube: source_bacteria_well.container.discard()
def pellet_bacteria(p, source_bacteria_well, antibiotic=None, destroy_source_tube=False, induce_high_copy_number=False): cell_line_name = get_cell_line_name(source_bacteria_well) assert isinstance(p,Protocol) if not antibiotic: #get the antibiotic from the source well well_antibiotic_str = source_bacteria_well.properties.get('antibiotic') if well_antibiotic_str: antibiotic = Antibiotic.from_string(well_antibiotic_str) else: raise Exception('Source Well must have property \'antibiotic\' set if antibiotic not specified') assert isinstance(antibiotic,Antibiotic) # Tubes and plates growth_plate = p.ref('growth_plate', cont_type="96-deep", discard=True) if induce_high_copy_number: overnight_volume = ul(550) growth_wells = growth_plate.wells_from(0,3,columnwise=True) else: #only use 50% to maximize growth overnight_volume = ul(1000) growth_wells = get_column_wells(growth_plate, range(0,4)) if destroy_source_tube: source_volume = get_volume(source_bacteria_well) - get_well_safe_volume(source_bacteria_well) else: source_volume = ul(50) #adding antibiotic mixes after by default p.add_antibiotic(growth_wells, antibiotic, broth_volume=overnight_volume) p.distribute(source_bacteria_well, growth_wells, round_volume(source_volume / len(growth_wells) * 1.0,2), allow_carryover=True) p.cover(growth_plate) #grow bacteria until they are in their log phase of growth #https://www.qiagen.com/us/resources/technologies/plasmid-resource-center/growth%20of%20bacterial%20cultures/ p.incubate(growth_plate, "warm_37", "{}:hour".format(16), shaking=True) p.uncover(growth_plate) if induce_high_copy_number: induction_wells = get_column_wells(growth_plate, 1) mix_well2 = p.ref('temp_mix_tube2', cont_type='micro-2.0', discard=True).well(0) overnight_bacteria_volume = ul(400) p.consolidate(growth_wells,mix_well2,floor_volume(overnight_bacteria_volume / len(growth_wells))) copy_control_volume = ul(2) #adding antibiotic mixes after by default p.add_antibiotic(mix_well2, antibiotic, total_volume_to_add_including_broth=ml(2)-overnight_bacteria_volume-\ copy_control_volume) p.provision_by_name(Reagent.copycontrol_induction_solution, mix_well2, copy_control_volume) p.distribute(mix_well2, induction_wells, floor_volume(get_volume(mix_well2,aspiratable=True) / len(induction_wells)), allow_carryover=True) p.incubate(growth_plate, "warm_37", "{}:hour".format(5), shaking=True) p.measure_bacterial_density(induction_wells) final_growth_wells = induction_wells else: final_growth_wells = growth_wells top_row_well_indices = range(0,8*4,8) top_wells = [growth_wells[i] for i in top_row_well_indices] non_top_wells = list(set(final_growth_wells) - set(top_wells)) p.measure_bacterial_density(top_wells, one_tip=True) #spin down each column of cells p.spin(growth_plate, '4000:g', '5:minute') trash_wells = growth_plate.wells_from(4,len(non_top_wells),columnwise=True) consolidate_volume = ul(250) #trash supernatent except consolidate_volume in non top row wells p.transfer(non_top_wells, trash_wells, get_volume(non_top_wells[0],aspiratable=True)-consolidate_volume, one_tip=True) #trash all volume in the top row wells trash_wells = growth_plate.wells_from(8,4,columnwise=True) p.transfer(top_wells, trash_wells, get_volume(top_wells[0],aspiratable=True), one_tip=True) #break up pellet in all wells p.mix(non_top_wells, repetitions=20, one_tip=True) #consolidate into the top row for top_well in top_wells: non_top_wells = set(growth_plate.wells_from(top_well.index,8,columnwise=True)) non_top_wells.remove(top_well) non_top_wells = list(non_top_wells) p.consolidate(non_top_wells, top_well, consolidate_volume) #spin down again and trash all but dead_volume+250ul p.spin(growth_plate, '4000:g', '5:minute') trash_wells = growth_plate.wells_from(9,4,columnwise=True) p.transfer(top_wells, trash_wells, get_volume(growth_wells[0],aspiratable=True)-consolidate_volume*2, one_tip=True) #break up the pellet created by spinning p.mix(top_wells, repetitions=20, one_tip=True) final_growth_wells = top_wells pellet_well = p.ref("pelleted_%s_cells"%(cell_line_name), cont_type="micro-2.0", storage="cold_80", cell_line_name=cell_line_name).well(0) copy_cell_line_name(source_bacteria_well, pellet_well) p.consolidate(final_growth_wells, pellet_well, get_volume(final_growth_wells[0],aspiratable=True), allow_carryover=True) p.measure_bacterial_density(pellet_well) p.spin(pellet_well.container, '4000:g', '10:minute') trash_well = p.ref('trash_tube', cont_type='micro-2.0', discard=True).well(0) #keep ul(25) to make the safe volume p.transfer(pellet_well, trash_well, pellet_well.volume-ul(25), one_source=True, one_tip=True) p.provision_by_name(Reagent.glycerol, pellet_well, ul(15),mix_after=True) #remove max (this means the remaining volume is 20% glycerol) p.transfer(pellet_well, trash_well, get_volume(pellet_well,aspiratable=True), one_source=True, one_tip=True) if destroy_source_tube: source_bacteria_well.container.discard()
def miniprep(p, source_bacteria_well, antibiotic=None, destroy_source_tube=False, incubate_hours=24, measure_od=True, induce_high_copy_number=False, slow_growth=False ): cell_line_name = get_cell_line_name(source_bacteria_well) assert isinstance(p,Protocol) if not antibiotic: #get the antibiotic from the source well well_antibiotic_str = source_bacteria_well.properties.get('antibiotic') if well_antibiotic_str: antibiotic = Antibiotic.from_string(well_antibiotic_str) else: raise Exception('Source Well must have property \'antibiotic\' set if antibiotic not specified') assert isinstance(antibiotic,Antibiotic) # Tubes and plates growth_plate = p.ref('growth_plate', cont_type="96-deep", discard=True) if slow_growth: growth_wells = get_column_wells(growth_plate,0) else: growth_wells = [growth_plate.well(0)] miniprep_result_plate = p.ref('%s_miniprep'%cell_line_name, cont_type="96-pcr", storage=Temperature.cold_4) miniprep_result_well = miniprep_result_plate.well(0) #adding antibiotic mixes after by default p.add_antibiotic(growth_wells, antibiotic, broth_volume=ul(1930)) source_bacteria_volume = ul(50)/len(growth_wells) p.transfer(source_bacteria_well,growth_wells,source_bacteria_volume,mix_before=True, mix_after=source_bacteria_volume<=ul(10)) interval_hr = 24 if incubate_hours>=24: for t in range(0,incubate_hours/interval_hr): p.incubate(growth_plate, "warm_37", "{}:hour".format(interval_hr), shaking=True) if measure_od: p.measure_bacterial_density(growth_wells, blanking_antibiotic=antibiotic, one_tip=True) if incubate_hours%24: p.incubate(growth_plate, "warm_37", "{}:hour".format(incubate_hours%24), shaking=True) if measure_od: p.measure_bacterial_density(growth_wells, blanking_antibiotic=antibiotic, one_tip=True) if induce_high_copy_number: induction_wells = growth_plate.wells_from(1,len(growth_wells),columnwise=True) overnight_bacteria_volume_to_use = ul(400) p.transfer(growth_wells, induction_wells, overnight_bacteria_volume_to_use, mix_before=True) #adding antibiotic mixes after by default p.add_antibiotic(induction_wells, antibiotic, total_volume_to_add_including_broth=ul(1998)-overnight_bacteria_volume_to_use) p.provision_by_name(Reagent.copycontrol_induction_solution, induction_wells, ul(2)) p.incubate(growth_plate, "warm_37", "{}:hour".format(5), shaking=True) if measure_od: p.measure_bacterial_density(induction_wells, blanking_antibiotic=antibiotic, one_tip=True) growth_wells = induction_wells if slow_growth: #consolidate bacteria trash_wells = get_column_wells(growth_plate,11) set_name(trash_wells,'trash_well') p.spin(growth_plate, '4000:g', '5:minute') consolidation_well = growth_wells[0] trash_volumes = [get_volume(consolidation_well, aspiratable=True)]+\ [get_volume(consolidation_well, aspiratable=True)-ul(298)]*(len(growth_wells)-1) p.transfer(growth_wells, trash_wells, trash_volumes) p.mix(growth_wells[1:],repetitions=20) p.consolidate(growth_wells[1:], consolidation_well,ul(283),allow_carryover=True, mix_after=True) if measure_od: p.measure_bacterial_density(consolidation_well, blanking_antibiotic=antibiotic, one_tip=True) final_growth_well = growth_wells[0] final_growth_well.name = miniprep_result_plate.name p.miniprep(final_growth_well,miniprep_result_well) p.measure_concentration(miniprep_result_well, "dna concentration", 'DNA') if destroy_source_tube: source_bacteria_well.container.discard()