def get_protocol(self):
p = stepwise.Protocol()
## Clean your bench
p += stepwise.load('rnasezap')
## In vitro transcription
rxn = self.reaction
n = plural(rxn.num_reactions)
f = self.footnotes.get('reaction', [])
p += paragraph_list(
f"Setup {n:# in vitro transcription reaction/s}{p.add_footnotes(*f)}:",
rxn,
unordered_list(*self.instructions),
)
f = self.footnotes.get('incubation', [])
if self.short and affected_by_short(self.incubation_times_min):
f += [
f"Reaction time is different than usual because the template is short (<{self.template_length_threshold} bp)."
]
p += f"Incubate at {self.incubation_temp_C}°C for {format_min(pick_by_short(self.incubation_times_min, self.short))}{p.add_footnotes(*f)}."
## DNase treatment
if self.dnase:
f = self.footnotes.get('dnase', [])
p += paragraph_list(
f"Setup {n:# DNase reaction/s}{p.add_footnotes(*f)}:",
self.dnase_reaction,
)
p += f"Incubate at {self.dnase_incubation_temp_C}°C for {format_min(self.dnase_incubation_time_min)}."
return p
def get_protocol(self):
anneal = AnnealMrnaLinker(self.mrnas, self.linkers)
anneal.num_reactions = self.num_reactions
anneal.linker_ratio = 0.6 # See expt #1
# Scale the reactions such that we'll end up with enough mRNA:
anneal_rxn = anneal.reaction
mrna_pmol = (
self.expected_dead_volume_uL * self.target_conc_uM /
self.expected_yield
)
mrna_conc_uM = anneal_rxn['mRNA'].stock_conc.value
anneal.mrna_volume_uL = max(
mrna_pmol / mrna_conc_uM,
self.mrna_volume_uL,
)
ligate = LigateMrnaLinker(anneal)
wash = WashBarendt()
wash.mwco_kDa = self.mwco_kDa
p = stepwise.Protocol()
p += anneal.protocol
p += ligate.protocol
if self.label:
p += f"Label the product: {self.label}"
if self.wash:
p += wash.protocol
if self.aliquot:
p += stepwise.load('aliquot "4 µL" "1 µM"')
return p
def get_protocol(self):
p = stepwise.Protocol()
if self.sample_mix:
p += self.prep_step
p += self.run_step
if self.stain:
p += stepwise.load(self.stain)
return p
def from_product(cls, product):
maker = cls()
args = product.maker_args
maker.products = [product]
if 'deps' in args:
maker.dependencies = {x.strip() for x in args['deps'].split(',')}
if 'seq' in args:
maker._product_seqs = [args['seq']]
if 'conc' in args:
maker._product_concs = [Quantity.from_string(args['conc'])]
if 'volume' in args:
maker._product_volumes = [Quantity.from_string(args['volume'])]
if 'molecule' in args:
maker._product_molecules = [args['molecule']]
if 'cwd' in args:
cwd = expanduser(args['cwd'])
elif 'expt' in args:
import exmemo
root = expanduser(args.get('project', getcwd()))
work = exmemo.Workspace.from_path(root)
expt = work.pick_experiment(args['expt'])
cwd = expt.root_dir.resolve()
else:
cwd = getcwd()
load_cmd = ' '.join(args.by_index[1:])
if not load_cmd:
raise QueryError("no stepwise command specified", culprit=product)
with cd(cwd):
maker.protocol = stepwise.load(load_cmd).protocol
maker._protocol_str = maker.protocol.format_text()
return maker
def get_protocol(self):
p = stepwise.Protocol()
footnotes = [x] if (x := self.protocol_link) else []
footnotes += self.footnotes
s = pl(f"Stain gel with {self.title}{p.add_footnotes(*footnotes)}:")
if self.light_sensitive:
#s += "Keep gel in the dark in all following steps."
s += ul("Keep the stain protected from light.")
#s += ul("In all following steps, protect the stain from light.")
s += self._format_stage('prep')
s += self._format_stage('stain')
s += self._format_stage('destain')
p += s
if self.imaging_cmd:
p += stepwise.load(self.imaging_cmd)
return p
#!/usr/bin/env python3
import stepwise
p = stepwise.Protocol()
p += "load"
p += stepwise.load("main")
p += stepwise.load("argv 1 2")
p += stepwise.load("import")
p.print()
p = stepwise.Protocol()
# Deciding how much to load:
# - I want one lane to be almost maximally concentrated, just to make sure I
# see something.
# - Normally I load 200 ng on urea gels, so I should have at least one lane
# close to that.
# - [Filonov2015] claims to be able to detect 100 pg of RNA (Figure 2), so that
# makes sense as a lower bound.
p += stepwise.load([
'serial',
'2 µL',
f'{db[mrna].conc_ng_uL} ng/µL',
0.1,
8,
'-m',
'f97',
'-d',
'nuclease-free water',
])
p += stepwise.load([
'gel',
'urea',
mrna,
'-S',
])
p += stepwise.load('stain_dfhbi')
p.print()
o127 400 µM 1 µL yes
PBS 2x 2 µL yes
""")
rxn.num_reactions = 4
p += f"""\
Setup 4 click reactions:
{rxn}
"""
p += """\
Incubate the reactions as follows:
- 25°C for 4h
- 25°C for 3h30, 65°C for 30 min
- 37°C for 4h
- 37°C for 3h30, 65°C for 30 min
"""
p += stepwise.load("gel anneal 6 -c 1990")
p.steps[2] = p.steps[2].replace(':', ' [1]:')
p.footnotes[1] = """\
The product (o129) has MW = 19899 g/mol, so:
100 µM = 1990 ng/µL
"""
print(p)
#!/usr/bin/env python3
import stepwise
import po4
p = stepwise.Protocol()
p += """\
In the following steps, setup these reactions:
Buffer: Mn Mn Mn Mn Mn Mn Mg Mg Mg Mg Mg Mg
dCas9: + − + − + + + − + − + +
DNA: − + + + + + − + + + + +
HUH-target: − − − + + + − − − + + +
EDTA: − − − − − + − − − − − +
"""
p += stepwise.load('huh/huh_cas9 f99 f100 -n 4 -S -b "Mn²⁺/Mg²⁺ buffer" -B 2.5')
p += stepwise.load('gel sdsmax 12 -S')
p += stepwise.load('gelgreen -r')
p += stepwise.load('simplyblue -fr')
print(p)
def rnase_digestion():
rxn = MasterMix("""\
Reagent Stock Volume
============= ====== ========
water to 10 µL
IVTT reaction 2.5 µL
EDTA 500 mM 1.25 µL
RNase A 1.0 µL
""")
return f"""\
Setup an RNase A digestion:
{rxn}
- Incubate at 60°C for 30 min.
"""
p = Protocol()
p += load('nebex p27 -v 12 -d 75/5')
p += hydroxide_hydrolysis()
p += carbonate_hydrolysis()
p += rnase_digestion()
p += load('gel sdsmax −lysate,−p27,−treatment,NaOH,NaHCO₃,RnaseA -S')
p += load('laser blue')
p += load('gelgreen -r')
print(p)
#!/usr/bin/env python3
import stepwise
from stepwise import pl, ul
p = stepwise.Protocol()
p += stepwise.load('cond optimize_click_freeze_cond.xlsx')
rxn = stepwise.MasterMix("""\
Reagent Stock Volume MM?
======= ====== ====== ===
PBS 2x 2 µL -
o126 400 µM 1 µL +
o233 400 µM 1 µL +
""")
rxn.num_reactions = 2
rxn.extra_percent = 0
rxn.hold_ratios.volume = '3 µL'
p += pl(
f"Setup {rxn.num_reactions} click reactions:",
rxn,
)
p += "Divide each reaction in three."
p += pl(
"Incubate the reactions as follows:",
ul(
"−20°C overnight",
"25°C for 4h, −20°C overnight",
p += f"""\
Make NM plates with the following additives:
{stepwise.tabulate(additives, header)}
"""
p += """\
Grow fresh colonies of s4 and s5 to OD≈1 in 3 mL
LB+Carb+Kan.
"""
p += """\
Wash cells with minimal media:
- Spin 3500g, 3 min, 4°C.
- Resuspend in 1 mL NM.
- Spin 3500g, 3 min, 4°C.
- Resuspend in 1 mL NM.
- Dilute to OD=0.1
"""
p += stepwise.load('serial 90µL 0.1OD 6 -f 10 -m cells -d NM')
p += """\
Plate 5 µL of each dilution in triplicate on each
plate.
"""
print(p)
#!/usr/bin/env python3
import stepwise
import wang2017
p = stepwise.Protocol()
p += """\
Prepare 100 U/mL RNase H:
- 44 µL nuclease-free water
- 5 µL 10x RNase H buffer
- 1 µL 5000 U/mL RNase H
"""
p += stepwise.load(
'serial 12µL 100U/mL 0.1 7 -m "RNase H" -d "1x RNase H reaction buffer"')
p.footnotes[2] = """\
If I could use an Echo, I would add 25 nL of 100 µM
o210/o211, to minimize the amount of volume added to
the sample. But I don't think I can accurately
pipet less than 0.5 µL by hand.
"""
p += """\
Prepare 5 µM o210-o212:
- 1 µL 100 µM oligo
- 19 µL nuclease-free water
"""
p += f"""\
Couple EMCS to the amine in the annealing oligo:
{emcs}
- Incubate at 37°C for 30 min [3].
"""
p.footnotes[3] = """\
Do not allow the solution to react for over 30
min because a longer reaction time causes
hydrolysis of the maleimide group in EMCS.
"""
p += stepwise.load('ethanol_precipitation')
p += """\
Couple the puromycin and annealing oligos:
- Resuspend the precipitated annealing oligo (now
coupled to EMCS) in the eluted puromycin oligo.
- Incubate at 4°C overnight.
- Add 1/20 volume 1M DTT.
- Incubate at room termperature for 30 min.
- Vortex periodically.
"""
p += """\
Perfrom another ethanol precipitation.
p += f"""\
Setup {plural(bal31.num_reactions):# BAL-31 digestion/s} [1]:
{bal31}
- Incubate at 30°C for 10 min.
- Add EGTA to 30 mM
- Incubate at 65°C for 10 min.
"""
p.footnotes[1] = """\
The NEB protocol calls for 3 pmol of DNA ends.
This reaction has 1 pmol DNA, which is 2 pmol
ends. Maybe I should use less BAL-31 to
compensate, but I think it's close enough.
"""
p.footnotes[2] = """\
This is the Cas9 reaction buffer recommended by
NEB. I might instead use whatever buffer Jorge
has been using, or another similar buffer based on
what I actually have.
"""
# 3x regular volume, for 3 technical replicates
# per reaction.
p += load(
"pcr dCas9-PCV2-DNA o87 o88 7 -a 65 -x 10 -p ssoadv -v 66 -m primers")
print(p)
huh = stepwise.MasterMix.from_text("""\
Reagent Stock Volume MM?
======== ====== ======== ===
water to 10 µL yes
CutSmart 10x 1 µL yes
dCas9 1 µM 1 µL yes
sgRNA 1 µM 1 µL yes
EDTA 500 mM 1 µL
HUH-DNA 200 nM 5 µL
""")
if __name__ == '__main__':
args = docopt.docopt(__doc__)
p = stepwise.Protocol()
p += stepwise.load('zap')
p += """\
In the following steps, setup these reactions:
dCas9: − + + + +
DNA (f12,f16): + − + + +
HUH-target: + − − + +
EDTA: − − − − +
"""
huh.num_reactions = 4
huh.extra_min_volume = '0.5 µL'
huh['water'].name = "nuclease-free water"
huh['dCas9'].name = "dCas9-PCV2 [1]"
huh['sgRNA'].name = "sgRNA (f13)"
- Add 20 mL 5M NaCl.
- Shake gently for 5 min.
3: Stain with GelGreen:
- Submerge gel in 3x GelGreen, 100 mM NaCl.
- Shake gently for 30 min.
4,5: Stain with Coomassie
- Submerge gel in SimplyBlue SafeStain.
- Microwave until almost boiling (45-60s).
- Shake gently for 5 min.
- Discard stain and add 100 mL water.
- Shake gently for 10 min.
- Add 20 mL 5M NaCl.
- Shake gently for 5 min.
"""
p.footnotes[1] = Footnote("""\
Stain the washed and unwashed gel pieces
separately, because it's possible that the excess
SDS from the unwashed gels could interfere with
staining.
""")
p += stepwise.load('laser blue red')
p.print()
huh['EDTA'].hold_stock_conc.conc = 30 / 2.5 * float(
args['--buffer-divalent-conc']), 'mM'
huh['DNA'].hold_conc.stock_conc = dna_nM, 'nM'
huh['DNA'].name = ','.join(dna)
mm = args['--master-mix'].split(',')
huh['dCas9-PCV2'].master_mix = 'cas9' in mm
huh['sgRNA'].master_mix = 'sgrna' in mm
huh['DNA'].master_mix = 'dna' in mm
huh['EDTA'].master_mix = 'edta' in mm
huh['buffer'].master_mix = 'buffer' in mm
if args['--no-sgrna']:
del huh['sgRNA']
else:
p += stepwise.load('zap')
huh['water'].name = 'nuclease-free water'
p += f"""\
Attach DNA to HUH-tagged dCas9 [1-3]:
{huh}
- Add each reagent in order.
- Mix after adding EDTA (and before adding DNA).
- Incubate at 37°C for 15 min.
"""
# 500 ng/band
p.footnotes[1] = """\
Invitrogen recommends loading no more than 250
