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()