def get_reaction(self):
rxn = stepwise.MasterMix()
rxn.volume = '20 µL'
rxn['T4 ligase buffer'].volume = '2.0 μL'
rxn['T4 ligase buffer'].stock_conc = '10x'
rxn['T4 ligase buffer'].master_mix = True
rxn['T4 ligase buffer'].order = 2
enz_uL = 0.5 if self.num_fragments <= 10 else 1.0
rxn['T4 DNA ligase'].volume = enz_uL, 'µL'
rxn['T4 DNA ligase'].stock_conc = '400 U/μL'
rxn['T4 DNA ligase'].master_mix = True
rxn['T4 DNA ligase'].order = 3
enzyme_db = NebRestrictionEnzymeDatabase()
for enzyme in self.enzymes:
stock = enzyme_db[enzyme]['concentration'] / 1000
rxn[enzyme].volume = enz_uL, 'µL'
rxn[enzyme].stock_conc = stock, 'U/µL'
rxn[enzyme].master_mix = True
rxn[enzyme].order = 4
return self._add_fragments_to_reaction(rxn)
def get_reaction(self):
rxn = stepwise.MasterMix()
rxn.num_reactions = n = self.num_reactions
rxn.solvent = None
rxn['mRNA'].stock_conc = consensus(
get_conc_uM(self.db, x, self.mrna_stock_uM) for x in self.mrnas)
rxn['linker'].stock_conc = consensus(
get_conc_uM(self.db, x, self.linker_stock_uM)
for x in self.linkers)
rxn['mRNA'].volume = self.mrna_volume_uL, 'µL'
rxn['linker'].volume = (
self.linker_ratio * rxn['mRNA'].volume *
(rxn['mRNA'].stock_conc / rxn['linker'].stock_conc))
rxn['mRNA'].master_mix = 'mrna' in self.master_mix
rxn['linker'].master_mix = 'link' in self.master_mix
if self.mrnas:
rxn['mRNA'].name = ','.join(self.mrnas)
if self.linkers:
rxn['linker'].name = ','.join(self.linkers)
rxn['PBS'].volume = rxn.volume / 9
rxn['PBS'].stock_conc = '10x'
rxn['PBS'].order = -1
return rxn
def elution_buffer():
return stepwise.MasterMix("""\
Reagent Stock Volume
=================== ====== ==========
nuclease-free water to 1000 µL
Tris, pH 7.5 1 M 10 µL
NaCl 5 M 100 µL
EDTA 500 mM 2 µL
SDS 10% 10 µL
""")
def get_reaction(self):
rxn = stepwise.MasterMix()
rxn.volume = '20 µL'
rxn['Gibson master mix'].volume = rxn.volume / 2
rxn['Gibson master mix'].stock_conc = "2x"
rxn['Gibson master mix'].catalog_num = 'NEB E2611'
rxn['Gibson master mix'].master_mix = True
rxn['Gibson master mix'].order = 2
return self._add_fragments_to_reaction(rxn)
def get_dnazyme_buffer(self):
rxn = stepwise.MasterMix()
rxn.volume = 20 * (self.num_reactions + 5), 'µL'
rxn.extra_min_volume = '1 µL'
rxn['Tris pH=8.5'].stock_conc = '1000 mM'
rxn['Tris pH=8.5'].hold_stock_conc.conc = '250 mM'
rxn['NaCl'].stock_conc = '5000 mM'
rxn['NaCl'].hold_stock_conc.conc = '500 mM'
rxn['MgCl₂'].stock_conc = '2000 mM'
rxn['MgCl₂'].hold_stock_conc.conc = '5 mM'
if rxn['MgCl₂'].volume < '1 µL':
rxn.hold_ratios.volume *= '1 µL' / rxn['MgCl₂'].volume
return rxn
def get_reaction(self):
rxn = stepwise.MasterMix()
rxn.volume = '100 µL'
rxn.num_reactions = self.num_reactions
rxn['water'].name = 'nuclease-free water'
rxn['DNAzyme buffer'].stock_conc = '5x'
rxn['DNAzyme buffer'].hold_stock_conc.conc = '1x'
rxn['DNAzyme buffer'].master_mix = True
rxn['o213'].stock_conc = '100 µM'
rxn['o213'].hold_stock_conc.conc = '0.1 µM'
rxn['o213'].master_mix = True
rxn['samples'].volume = '10.5 µL'
rxn['samples'].master_mix = False
while rxn['o213'].volume * rxn.scale < '0.5 µL':
rxn['o213'].hold_conc.stock_conc /= 2
return rxn
def get_reaction(self):
rxn = stepwise.MasterMix("""\
Reagent Stock Volume MM?
============================= ========= ======== ===
water to 50 µL +
S30 extract [2] 12 µL +
synthesis buffer [2] 2x 25 µL +
T7 RNA polymerase 450 U/µL 1 µL +
RNase inhibitor (murine) 40 U/µL 1 µL +
GamS nuclease inhibitor [2,3] 1.5 µg/µL 1 µL +
""")
rxn.hold_ratios.volume = self.volume_uL, 'µL'
rxn.num_reactions = self.num_reactions or len(self.templates)
rxn['template'].name = f"{','.join(self.templates)} [3]"
rxn['template'].stock_conc = self.template_stock_nM, 'nM'
rxn['template'].hold_stock_conc.conc = self.template_conc_nM, 'nM'
rxn.hold_ratios.volume = self.volume_uL, 'µL'
if self.use_mrna:
del rxn['T7 RNA polymerase']
return rxn
#!/usr/bin/env python3
import docopt
import stepwise
import wang2017
controls = stepwise.MasterMix("""\
Reagent Stock Volume MM?
=============================== ======= ========== ===
nuclease-free water to 10.5 µL x
o210,o211,o212 5 µM 0.5 µL
""")
purex = stepwise.MasterMix("""\
Reagent Stock Volume MM?
=============================== ======= ========== ===
nuclease-free water to 10.5 µL x
A 4.0 µL x
B 3.0 µL x
RNase inhibitor (murine) 40 U/µL 0.2 µL x
o210,o211,o212 5 µM 0.5 µL
""")
pfrex = stepwise.MasterMix("""\
Reagent Stock Volume MM?
=============================== ======= ========== ===
nuclease-free water to 10.5 µL x
Solution I 5.0 µL x
Solution II 0.5 µL x
Solution III 1.0 µL x
o210,o211,o212 5 µM 0.5 µL
def get_reactions(self):
# Define the annealing reaction:
anneal = stepwise.MasterMix()
anneal.num_reactions = self.num_reactions
anneal.solvent = None
anneal.extra_min_volume = 0.5, 'µL'
template_fmol = self.volume_uL * self.template_conc_nM
anneal['template'].name = ','.join(self.templates)
anneal['template'].stock_conc = self.template_stock_nM, 'nM'
anneal[
'template'].volume = template_fmol / self.template_stock_nM, 'µL'
anneal['template'].master_mix = 'rna' in self.master_mix
anneal['template'].order = 2
anneal['primer'].name = ','.join(self.primers)
anneal['primer'].stock_conc = self.primer_stock_uM, 'µM'
anneal[
'primer'].volume = self.primer_excess * template_fmol / self.primer_stock_uM / 1e3, 'µL'
anneal['primer'].master_mix = 'primer' in self.master_mix
anneal['primer'].order = 3
# If necessary, dilute the annealing reaction such that it will be
# necessary to add the given volume to the RT reaction. The purpose of
# this is to ensure that we can pipet this volume accurately, since we
# often want to be quantitative about how much RNA we have (e.g. qPCR).
if anneal.volume < (self.rna_min_volume_uL, 'µL'):
anneal.solvent = 'nuclease-free water'
anneal.volume = self.rna_min_volume_uL, 'µL'
anneal['first-strand buffer'].stock_conc = '5x'
anneal['first-strand buffer'].volume = 0, 'µL'
anneal['first-strand buffer'].volume = anneal.volume / 4
anneal['first-strand buffer'].master_mix = bool(self.master_mix)
anneal['first-strand buffer'].order = 1
# Define the MMLV reaction:
mmlv = stepwise.MasterMix("""\
Reagent Stock Volume MM?
======================== ======== ========== ===
nuclease-free water to 20.0 µL yes
first-strand buffer 5x 4.0 µL yes
dNTP mix 10 mM 2.0 µL yes
DTT 100 mM 2.0 µL yes
SMART MMLV RT 200 U/µL 0.5 µL yes
annealed template/primer 0.0 µL
""")
mmlv.num_reactions = self.num_reactions
mmlv.hold_ratios.volume = self.volume_uL, 'µL'
mmlv['first-strand buffer'].volume -= anneal[
'first-strand buffer'].volume
mmlv['annealed template/primer'].volume = anneal.volume
mmlv['annealed template/primer'].stock_conc = \
anneal['template'].stock_conc * (
anneal['template'].volume / anneal.volume)
# Scale the volume of the annealing reaction to guarantee that none of
# the volume are too small to pipet accurately.
min_pipet_volumes = {
'template': (self.rna_min_volume_uL, 'µL'),
}
pipet_volumes = [
(anneal.master_mix_volume, '0.5 µL'),
]
pipet_volumes += [
(anneal[k].volume, min_pipet_volumes.get(k, '0.5 µL'))
for k in ['template', 'primer'] if not anneal[k].master_mix
]
anneal.hold_ratios.volume *= max(
1 + self.extra_anneal_percent / 100,
*(limit / curr for curr, limit in pipet_volumes),
)
return anneal, mmlv
#!/usr/bin/env python3
import stepwise
p = stepwise.Protocol()
rxn = stepwise.MasterMix("""\
Reagent Stock Volume MM?
======= ====== ====== ===
o126 400 µM 1 µL yes
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")
#!/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",
later step).
"""
import docopt
import stepwise
args = docopt.docopt(__doc__)
# PBS:
# - I don't remember why I decided to add PBS to this reaction.
# - The Glen Research protocol referenced by expt #57 doesn't call for any
# salt.
rxn = stepwise.MasterMix("""\
Reagent Stock Volume
====================== ====== ======
puromycin oligo 400 µM 1 µL
annealing oligo 400 µM 1 µL
PBS 2x 2 µL
""")
rxn['puromycin oligo'].name = args['<puro-oligo>']
rxn['annealing oligo'].name = args['<anneal-oligo>']
if v := args['--volume']:
rxn.hold_ratios.volume = v, 'µL'
p = stepwise.Protocol()
p += stepwise.Step(
"Couple the linker oligos:",
rxn,
complete description of the syntax. This optiona can be specified
multiple times.
"""
import docopt
import stepwise
from inform import plural
args = docopt.docopt(__doc__)
protocol = stepwise.Protocol()
purexpress = stepwise.MasterMix('''\
Reagent Stock Volume MM?
=================== ========= ========== ===
water to 10.0 µL yes
A 4.0 µL yes
B 3.0 µL yes
RNase inhibitor [1] 40 U/µL 0.2 µL yes
ZnOAc 1 mM 0.5 µL yes
target DNA 750 nM 0.8 µL yes
template DNA 75 nM 0.8 µL
template mRNA 1000 nM 1.6 µL
''')
if not args['--add-zinc']:
del purexpress['ZnOAc']
if not args['--add-target']:
del purexpress['target DNA']
if args['--no-inhibitor']:
del purexpress['RNase inhibitor [1]']
else:
def get_protocol(self):
p = stepwise.Protocol()
if self.dnase_treatment == 'pre':
mm = stepwise.MasterMix("""\
Reagent Stock Volume MM?
====================== ======= ======== ===
nuclease-free water to 50 µL +
DNA digestion buffer 10x 5 µL +
DNase I [1] 1 U/µL 5 µL +
RNA sample <10 µg 40 µL -
""")
step = "Setup a DNase I reaction for each sample:"
if self.num_samples:
step = f"Setup {plural(self.num_samples):# DNase I reaction/s}:"
mm.num_reactions = self.num_samples
if self.sample_volume_uL:
mm['RNA sample'].volume = self.sample_volume_uL, 'µL'
p += pl(step, mm)
p += "Incubate at room temperature for 15 min."
p.footnotes[
1] = "Reconstitute lyophilized DNase I (#E1009-A; 250U) with 275 µL nuclease-free water and mix by gentle inversion. Store frozen aliquots."
if self.dnase_treatment == 'post':
mm = stepwise.MasterMix("""\
Reagent Stock Volume MM?
====================== ======= ======== ===
DNA digestion buffer 10x 75 µL +
DNase I [1] 1 U/µL 5 µL +
""")
if self.num_samples:
mm.num_reactions = self.num_samples
p += pl("Prepare 80 µL DNase I reaction mix for each sample:", mm)
s = ul()
p += pl("Purify RNA using Zymo Clean & Concentrator spin columns:", s)
if self.sample_volume_uL:
if self.sample_volume_uL < 50:
s += f"Add {50 - self.sample_volume_uL:g} nuclease-free water to each sample"
v = max(50, self.sample_volume_uL)
s += f"Add {2*v:g} µL RNA binding buffer; mix."
s += f"Add {3*v:g} µL >95% ethanol; mix."
else:
s += "If necessary, bring each sample to 50 µL."
s += "Add 2 volumes RNA binding buffer; mix."
s += "Add 3 volumes >95% ethanol; mix."
s += f"Load sample on spin column."
s += f"Spin 1 min, 16000g; discard flow-through."
if self.dnase_treatment == 'post':
s += f"Add 400 µL RNA wash buffer."
s += f"Spin 30s, 16000g; discard flow-through."
s += f"Add 80 µL DNase I reaction mix."
s += f"Incubate at room temperature for 15 min."
s += f"Add 400 µL RNA prep buffer."
s += f"Spin 30s, 16000g; discard flow-through."
s += f"Add 700 µL RNA wash buffer."
s += f"Spin 30s, 16000g; discard flow-through."
s += f"Add 400 µL RNA wash buffer."
s += f"Spin 60s, 16000g; discard flow-through."
s += f"Add {self.elute_volume_uL:g} µL nuclease-free water."
s += f"Spin 30s, 16000g."
return p
def get_reaction_neb(self):
# Enzyme volume:
# - One unit is defined as the amount of enzyme required to convert 1
# nanomole of 5´-[32P]rA16 into a phosphatase-resistant form in 30
# minutes at 37°C.
#
# - Compared to the unit definition for the Takara enzyme, this
# definition has:
#
# - 1000x more RNA
# - 3x longer reaction time
# - 28°C warner reaction temperature
#
# - The NEB enzyme is also 10 U/µL, which the Takara one is 40 U/µL.
#
# - [Reyes2021] used:
# - 20 U T4 RNA ligase
# - 3 U T4 PNK
# - 400 pmol mRNA (100 µL at 4 µM)
#
# Scaled to 5 pmol RNA (40 µL at 125 nM):
# - 0.25 U (0.025 µL) T4 RNA ligase
# - 0.0375 U (0.00375 µL) T4 PNK
#
# - The NEB ligation protocol calls for:
# - 10 U T4 RNA ligase
# - 20 pmol RNA
# - 2h incubation at 25°C
# - https://www.neb.com/protocols/2018/10/17/protocol-ligation-of-an-oligo-to-the-3-end-of-rna-using-t4-rna-ligase-1m0204
#
# Scaled to 5 pmol RNA (40 µL at 125 nM):
# - 2.5 U T4 RNA ligase (0.25 µL)
#
# - I'm going to follow [Reyes2021]:
#
# - [Reyes2021] uses annealed oligos, like my reaction. The other
# reactions/definitions are based on unannealed RNAs. It's very
# likely that the ligation reaction would be much faster with
# annealed oligos, meaning that less enzyme would be needed.
#
# - The reactions I'm doing are big enough that if I don't follow
# [Reyes2021], I'd need to use ≈10 µL volumes of enzyme, which
# would get very expensive. Maybe I'll need to optimize this, but
# for now it just feels more practical to use the smaller volumes
# called for by [Reyes2021].
#
# - I used the T4 PNK concentration from [Reyes2021], even though I
# normally don't use PNK. As with the ligase, this PNK
# concentration is also much lower than what I'd been using in the
# Takara reaction.
#
# - Update: 2021/05/06
#
# - I decided to double the enzyme volumes relative to [Reyes2021]_,
# because I suspect that I wasn't getting as much ligation with
# those volumes as I used to. I haven't rigorously tried to
# optimize this, yet.
rxn = stepwise.MasterMix("""\
Reagent Stock Volume MM?
===================== ======= =========== ===
nuclease-free water to 40.00 µL +
T4 RNA ligase buffer 10x 4.0 µL +
ATP 10 mM 4.0 µL +
PEG 8000 50% 20.0 µL +
T4 PNK 10 U/µL 0.008 µL -
T4 RNA ligase 10 U/µL 0.050 µL -
annealed mRNA/linker 1.25 µM 4.0 µL -
""")
rxn['T4 RNA ligase'].name = "T4 RNA ligase (NEB M0204)"
return self._adjust_reaction(rxn)
args = docopt.docopt(__doc__)
rxn_uL = float(args['--volume'])
mg_mM = float(args['--mg-conc'])
mg_stock_mM = float(args['--mg-stock'])
k_mM = float(args['--k-conc'])
k_stock_mM = float(args['--k-stock'])
M = np.array([
[mg_mM, -mg_stock_mM, 0, 0],
[ k_mM, 0, -k_stock_mM, 0],
[ 1, -1, -1, rxn_uL],
])
salt_uL = np.linalg.solve(M[:,:3], M[:,3])
rxn = stepwise.MasterMix()
rxn.num_reactions = int(args['--num-reactions'])
rxn.extra_min_volume = 5, 'µL'
rxn.solvent = None
rxn['translation reaction'].volume = rxn_uL, 'µL'
rxn['translation reaction'].name = args['<reagent>']
rxn['translation reaction'].stock_conc = args['--ivtt-stock']
rxn['MgOAc'].stock_conc = mg_stock_mM, 'mM'
rxn['MgOAc'].volume = salt_uL[1], 'µL'
rxn['MgOAc'].master_mix = True
rxn['KCl'].stock_conc = k_stock_mM, 'mM'
rxn['KCl'].volume = salt_uL[2], 'µL'
rxn['KCl'].master_mix = True
p = stepwise.Protocol()
p += pl(
primer_scale = pcr.scale if pcr[p].master_mix else 1
primer_vol = primer_scale * pcr[p].volume
if primer_vol < '0.5 µL':
use_primer_mix.append(p)
if use_primer_mix:
pcr['primer mix'].order = 4
pcr['primer mix'].stock_conc = '10x'
pcr['primer mix'].volume = pcr.volume / 10
pcr['primer mix'].master_mix = all(
pcr[p].master_mix
for p in use_primer_mix
)
primer_mix = stepwise.MasterMix()
primer_mix.volume = pcr.volume
for p in use_primer_mix:
primer_mix[p].name = pcr[p].name
primer_mix[p].stock_conc = pcr[p].stock_conc
primer_mix[p].volume = pcr[p].volume
primer_mix[p].hold_stock_conc.conc *= 10
del pcr[p]
primer_mix.hold_ratios.volume = '10 µL'
return pcr, primer_mix
def get_templates(self):
return [x.template for x in self.amplicons]
