def test_total_plate_volume(self):
plate = create_blank_plate('24-deep')
plate.well(0).volume = ml(10)
plate.well(1).volume = ml(5) + ul(30)
self.assertEqual(utils.total_plate_volume(plate, aspiratable=True),
ml(15))
self.assertEqual(utils.total_plate_volume(plate, aspiratable=False),
ml(15) + ul(30))
def test_breakup_dispense_column_volumes(self):
col_vol_pairs = [{
"column": 0,
"volume": ml(4)
}, {
"column": 1,
"volume": ml(5)
}, {
"column": 2,
"volume": ml(2)
}]
self.assertListEqual([{
"column": 0,
"volume": ml(2)
}, {
"column": 0,
"volume": ml(2)
}, {
"column": 1,
"volume": ml(2.5)
}, {
"column": 1,
"volume": ml(2.5)
}, {
"column": 2,
"volume": ml(2)
}], utils.breakup_dispense_column_volumes(col_vol_pairs))
def test_convert_mass_to_volume(self):
plate = create_blank_plate('24-deep')
dna_well = plate.well(0)
dna_well.volume = ml(10)
dna_well.properties['Concentration (DNA)'] = '4:nanograms/microliter'
self.assertEqual(utils.convert_mass_to_volume(utils.ng(20), dna_well),
ul(5))
def test_convert_moles_to_volume(self):
plate = create_blank_plate('24-deep')
dna_well = plate.well(0)
dna_well.volume = ml(10)
dna_well.properties['Concentration (DNA)'] = '649:nanograms/microliter'
dna_well.properties['dna_length'] = 1000
self.assertEqual(
utils.convert_moles_to_volume(utils.pmol(1), dna_well), ul(1))
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 test_available_plate_volume(self):
plate = create_blank_plate('24-deep')
self.assertEqual(utils.space_available(plate), ml(10) * 24)