for iteration in range(2):
print "Iteration ",iteration+1
trp=TRP()
if iteration==0:
trp.addTemplates(inputs,tmplConc*1e9) # Add a template with stock concentration
else:
reagents=Sample.getAllOnPlate(Experiment.REAGENTPLATE)
for r in reagents:
if r.volume<0:
r.initvolume=-r.volume+20
Sample.clearall()
# Round 1 (Keep cleaved -theo)
print "***** Round 1 T7 *****"
t71=trp.runT7New(theo=[False for i in inputs]+[True for i in inputs],src=inputs+inputs,tgt=[],vol=10,srcdil=10.0/3)
sv1t7=trp.saveSamps(src=t71,vol=3,dil=25,plate=trp.e.DILPLATE)
rt1=trp.runRT(pos=True,src=t71,tgt=[],vol=5,srcdil=2)
trp.diluteInPlace(tgt=rt1,dil=4)
lig1=trp.runLig(prefix="B",src=rt1,tgt=[],vol=10,srcdil=3)
trp.diluteInPlace(tgt=lig1,dil=5)
# Save in dilution plate
sv1lig=trp.saveSamps(src=lig1,vol=20,dil=5,plate=trp.e.DILPLATE)
# Only need to PCR -theo case, cleaved
pcr1=trp.runPCR(prefix="B",src=lig1[0:len(inputs)],tgt=["%s.c"%i for i in inputs],vol=25,srcdil=4,ncycles=cycles)
trp.diluteInPlace(tgt=pcr1,dil=3)
sv1pcr=trp.saveSamps(src=pcr1,tgt=[], vol=50,dil=1)
trp = TRP()
if iteration == 0:
trp.addTemplates(inputs, tmplConc *
1e9) # Add a template with stock concentration
else:
reagents = Sample.getAllOnPlate(Experiment.REAGENTPLATE)
for r in reagents:
if r.volume < 0:
r.initvolume = -r.volume + 20
Sample.clearall()
# Round 1 (Keep cleaved -theo)
print "***** Round 1 T7 *****"
t71 = trp.runT7New(theo=[False for i in inputs] + [True for i in inputs],
src=inputs + inputs,
tgt=[],
vol=10,
srcdil=10.0 / 3)
sv1t7 = trp.saveSamps(src=t71, vol=3, dil=25, plate=trp.e.DILPLATE)
rt1 = trp.runRT(pos=True, src=t71, tgt=[], vol=5, srcdil=2)
trp.diluteInPlace(tgt=rt1, dil=4)
lig1 = trp.runLig(prefix="B", src=rt1, tgt=[], vol=10, srcdil=3)
trp.diluteInPlace(tgt=lig1, dil=5)
# Save in dilution plate
sv1lig = trp.saveSamps(src=lig1, vol=20, dil=5, plate=trp.e.DILPLATE)
# Only need to PCR -theo case, cleaved
pcr1 = trp.runPCR(prefix="B",
print "Iteration ", iteration + 1
trp = TRP()
if iteration == 0:
trp.addTemplates(inputs, tmplConc *
1e9) # Add a template with stock concentration
else:
reagents = Sample.getAllOnPlate(Experiment.REAGENTPLATE)
for r in reagents:
if r.volume < 0:
r.initvolume = -r.volume + 20
Sample.clearall()
# Round 1 (Keep uncleaved +theo)
t71 = trp.runT7New(theo=True,
src=inputs,
tgt=["%s.T1+" % i for i in inputs],
vol=10,
srcdil=10.0 / 3,
dur=30)
trp.diluteInPlace(tgt=t71, dil=5)
rt1 = trp.runRT(pos=True, src=t71, tgt=[], vol=5, srcdil=2)
trp.diluteInPlace(tgt=rt1, dil=4)
pcr1 = trp.runPCR(prefix=srcprefix, src=rt1, tgt=[], vol=25, srcdil=4)
trp.diluteInPlace(tgt=pcr1, dil=3)
sv1pcr = trp.saveSamps(src=pcr1,
tgt=["%s.R1" % i for i in inputs],
vol=55,
dil=1)
# Round 2 (both rounds, +/-theo, Ligate, keep cleaved)
r2in = sv1pcr + inputs
t72 = trp.runT7New(theo=[False for i in r2in] + [True for i in r2in],
# nM
for iteration in range(2):
print "Iteration ", iteration + 1
trp = TRP()
if iteration == 0:
trp.addTemplates(inputs, tmplConc * 1e9) # Add a template with stock concentration
else:
reagents = Sample.getAllOnPlate(Experiment.REAGENTPLATE)
for r in reagents:
if r.volume < 0:
r.initvolume = -r.volume + 20
Sample.clearall()
# Round 1 (Keep uncleaved +theo)
t71 = trp.runT7New(theo=True, src=inputs, tgt=["%s.T1+" % i for i in inputs], vol=10, srcdil=10.0 / 3, dur=30)
trp.diluteInPlace(tgt=t71, dil=5)
rt1 = trp.runRT(pos=True, src=t71, tgt=[], vol=5, srcdil=2)
trp.diluteInPlace(tgt=rt1, dil=4)
rtconc = tmplConc * 3.0 / 10.0 * rnagain / (2 * 5 * 2 * 4 * 4) # Expected concentration of ligation product here
cycles = math.ceil(math.log(endconc / rtconc, pcreff))
# Amplify to end of exponential phase
print "PCR input conc=%.3g pM, PCR cycles=%.1f, End Conc=%.0f nM" % (rtconc * 1e12, cycles, endconc * 1e9)
pcr1 = trp.runPCR(prefix=srcprefix, src=rt1, tgt=[], vol=25, srcdil=4, ncycles=cycles)
trp.diluteInPlace(tgt=pcr1, dil=3)
sv1pcr = trp.saveSamps(src=pcr1, tgt=["%s.R1" % i for i in inputs], vol=55, dil=1)
# Round 2 (both rounds, +/-theo, Ligate, keep cleaved)
r2in = sv1pcr + inputs
