def test_pcr_master_mix_components():
pcr = dirty_water.Pcr()
pcr.template_in_master_mix = False
pcr.primers_in_master_mix = False
assert not pcr.template_in_master_mix
assert not pcr.primers_in_master_mix
assert pcr.steps[0] == '''\
Setup 1 PCR reaction and 1 negative control:
Reagent Conc Each Rxn Master Mix
──────────────────────────────────────────────
Q5 master mix 2x 25.00 μL 50.00 μL
primer mix 10x 5.00 μL
template DNA 100 pg/μL 1.00 μL
water 19.00 μL 38.00 μL
──────────────────────────────────────────────
50.00 μL 44.00 μL/rxn'''
pcr.template_in_master_mix = True
pcr.primers_in_master_mix = False
assert pcr.template_in_master_mix
assert not pcr.primers_in_master_mix
assert pcr.steps[0] == '''\
Setup 1 PCR reaction and 1 negative control:
Reagent Conc Each Rxn Master Mix
──────────────────────────────────────────────
Q5 master mix 2x 25.00 μL 50.00 μL
primer mix 10x 5.00 μL
template DNA 100 pg/μL 1.00 μL 2.00 μL
water 19.00 μL 38.00 μL
──────────────────────────────────────────────
50.00 μL 45.00 μL/rxn'''
pcr.template_in_master_mix = False
pcr.primers_in_master_mix = True
assert not pcr.template_in_master_mix
assert pcr.primers_in_master_mix
assert pcr.steps[0] == '''\
Setup 1 PCR reaction and 1 negative control:
Reagent Conc Each Rxn Master Mix
──────────────────────────────────────────────
Q5 master mix 2x 25.00 μL 50.00 μL
primer mix 10x 5.00 μL 10.00 μL
template DNA 100 pg/μL 1.00 μL
water 19.00 μL 38.00 μL
──────────────────────────────────────────────
50.00 μL 49.00 μL/rxn'''
pcr.template_in_master_mix = True
pcr.primers_in_master_mix = True
assert pcr.template_in_master_mix
assert pcr.primers_in_master_mix
assert pcr.steps[0] == '''\
def test_pcr_num_reactions():
pcr = dirty_water.Pcr()
pcr.num_reactions = 2
assert pcr.num_reactions == 2
assert pcr.steps[0] == '''\
Setup 2 PCR reactions and 1 negative control:
Reagent Conc Each Rxn Master Mix
──────────────────────────────────────────────
Q5 master mix 2x 25.00 μL 75.00 μL
primer mix 10x 5.00 μL
template DNA 100 pg/μL 1.00 μL 3.00 μL
water 19.00 μL 57.00 μL
──────────────────────────────────────────────
50.00 μL 45.00 μL/rxn'''
pcr.num_reactions = 3
assert pcr.num_reactions == 3
assert pcr.steps[0] == '''\
def test_pcr_default_args():
pcr = dirty_water.Pcr()
assert pcr.num_reactions == 1
assert pcr.extra_master_mix == 0
assert pcr.template_in_master_mix == True
assert pcr.primers_in_master_mix == False
assert pcr.annealing_temp == 60
assert pcr.extension_time == 120
assert pcr.num_cycles == 35
assert pcr.steps[0] == '''\
Setup 1 PCR reaction and 1 negative control:
Reagent Conc Each Rxn Master Mix
──────────────────────────────────────────────
Q5 master mix 2x 25.00 μL 50.00 μL
primer mix 10x 5.00 μL
template DNA 100 pg/μL 1.00 μL 2.00 μL
water 19.00 μL 38.00 μL
──────────────────────────────────────────────
50.00 μL 45.00 μL/rxn'''
assert pcr.steps[1] == '''\
def test_pcr_extension_time():
pcr = dirty_water.Pcr()
pcr.extension_time = 59
assert pcr.steps[1] == '''\
Run the following thermocycler protocol:
98°C → 98°C → 60°C → 72°C → 72°C → 12°C
0:30 0:10 0:20 0:59 2:00 ∞
└──────────────────┘
35x'''
pcr.extension_time = 60
assert pcr.steps[1] == '''\
Run the following thermocycler protocol:
98°C → 98°C → 60°C → 72°C → 72°C → 12°C
0:30 0:10 0:20 1:00 2:00 ∞
└──────────────────┘
35x'''
pcr.extension_time = 61
assert pcr.steps[1] == '''\
def test_pcr_extra_master_mix():
pcr = dirty_water.Pcr()
pcr.extra_master_mix = 10
assert pcr.extra_master_mix == 10
assert pcr.steps[0] == '''\
def test_pcr_num_cycles():
pcr = dirty_water.Pcr()
pcr.num_cycles = 25
assert pcr.steps[1] == '''\
def test_pcr_annealing_temp():
pcr = dirty_water.Pcr()
pcr.annealing_temp = 62
assert pcr.steps[1] == '''\
- You can also order HPLC/PAGE purification if
your primers are long and you don't mind the
extra expense, but the regular purification
worked well for me."""
## Inverse PCR
def time_to_secs(time): #
if ':' in time:
min, sec = map(int, time.split(':'))
return 60 * min + sec
else:
# Assume the time is just a number of seconds.
return int(time)
pcr = dirty_water.Pcr()
pcr.num_reactions = num
pcr.annealing_temp = args['<annealing_temp>']
pcr.extension_time = time_to_secs(args['--extension-time'])
pcr.make_primer_mix = True
pcr.reaction.volume = eval(args['--reaction-volume'])
pcr.primer_mix['forward primer'].stock_conc = float(args['--primer-conc'])
pcr.primer_mix['reverse primer'].stock_conc = float(args['--primer-conc'])
protocol += pcr
## DpnI digestion
protocol += """\
Add 1 μL (20 units) DpnI to each reaction and
incubate at 37°C for 1h."""