trp.runT7Setup(theo=False,src=srcs,tgt=t71master,vol=len(timepoints)*12.2+15,srcdil=10.0/6)
t71=[];
for i in range(len(timepoints)):
t71=t71+trp.saveSamps(src=t71master,tgt=["%s.T%d"%(s,timepoints[i]) for s in srcs],vol=10,dil=1,plate=trp.e.SAMPLEPLATE)
# Stop one sample immediately for a zero timepoint
trp.runT7Stop(theo=False,vol=10, tgt=t71[0:len(srcs)],stopmaster=stop)
trp.runT7Pgm(vol=10,dur=timepoints[-1])
for s in findsamps(t71[len(srcs):]):
s.volume=s.volume+10 # Program doesn't know that they were diluted 2x by hand
s.addhistory("Manual stop",10,None)
# No stop, done during run
trp.diluteInPlace(tgt=t71,dil=5)
# Dilute input samples enough to use in qPCR directly (should be 5000/(rnagain*2*5) = 20)
qpcrdil1=trp.runQPCRDIL(src=t71,tgt=[],vol=100,srcdil=20,dilPlate=True)
rt1=trp.runRT(pos=True,src=t71,tgt=[],vol=5,srcdil=2)
rt1=trp.diluteInPlace(tgt=rt1,dil=5)
lig1=trp.runLig(src=rt1,tgt=[],vol=10,srcdil=3,master=ligmaster)
prods=trp.diluteInPlace(tgt=lig1,dil=10)
for i in range(len(qpcrdil1)):
trp.runQPCR(src=qpcrdil1[i],vol=15,srcdil=10.0/4,primers=[tmplqpcr[i]])
for i in range(len(prods)):
trp.runQPCR(src=[prods[i]],vol=15,srcdil=10.0/4,primers=pcr[i])
trp.finish()
trp.addTemplates(input,stockconc=10.0/6.0,units="x",plate=Experiment.EPPENDORFS) # Add a template
else:
reagents=Sample.getAllOnPlate(Experiment.REAGENTPLATE)+Sample.getAllOnPlate(Experiment.EPPENDORFS)
for r in reagents:
if r.volume<=0:
r.initvolume=-r.volume+r.plate.unusableVolume
Sample.clearall()
t71=trp.runT7(theo=False,src=srcs,tgt=[],vol=10,srcdil=10.0/6,dur=15,stopmaster=stop)
#print t71
t71=trp.diluteInPlace(tgt=t71,dil=5)
# Dilute input samples enough to use in qPCR directly (should be 5000/(rnagain*2*5) = 20)
qpcrdil1=trp.runQPCRDIL(src=t71,tgt=[],vol=100,srcdil=20,dilPlate=False)
rt1=trp.runRT(pos=True,src=t71,tgt=[],vol=30,srcdil=2)
rt1=trp.diluteInPlace(tgt=rt1,dil=5)
lig=trp.runLig(src=rt1*6,tgt=[],vol=20,srcdil=3,master=ligmaster1*3+ligmaster2*3)
prods=trp.diluteInPlace(tgt=lig,dil=10)
print "prod=",prods
for i in range(len(qpcrdil1)):
trp.runQPCR(src=qpcrdil1[i],vol=15,srcdil=10.0/4,primers=[tmplqpcr[i]],nreplicates=3)
for p in pcr1:
trp.runQPCR(src=prods[0:3],vol=15,srcdil=10.0/4,primers=p,nreplicates=3)
for p in pcr2:
trp.runQPCR(src=prods[3:6],vol=15,srcdil=10.0/4,primers=p,nreplicates=3)
trp.finish()
t71 = trp.runT7(theo=False,
src=srcs,
tgt=[],
vol=10,
srcdil=10.0 / 6,
dur=15,
stopmaster=stop)
#print t71
t71 = trp.diluteInPlace(tgt=t71, dil=5)
# Dilute input samples enough to use in qPCR directly (should be 5000/(rnagain*2*5) = 20)
qpcrdil1 = trp.runQPCRDIL(src=t71,
tgt=[],
vol=100,
srcdil=20,
dilPlate=True)
rt1 = trp.runRT(pos=True, src=t71, tgt=[], vol=5, srcdil=2)
rt1 = trp.diluteInPlace(tgt=rt1, dil=5)
lig1 = trp.runLig(src=rt1, tgt=[], vol=10, srcdil=3, master=ligmaster)
prods = trp.diluteInPlace(tgt=lig1, dil=10)
for i in range(len(qpcrdil1)):
trp.runQPCR(src=qpcrdil1[i],
vol=15,
srcdil=10.0 / 4,
primers=[tmplqpcr[i]])
for i in range(len(prods)):
trp.runQPCR(src=[prods[i]], vol=15, srcdil=10.0 / 4, primers=pcr[i])
trp.finish()
pcr1=trp.runPCR(prefix=[srcprefix],src=rt1[1],tgt=[],vol=50,srcdil=4)
trp.diluteInPlace(tgt=pcr1,dil=3)
sv1pcr=trp.saveSamps(src=pcr1,tgt=["R1"],vol=125,dil=1)
# Round 2 (-theo, Ligate, keep cleaved)
t72=trp.runT7(theo=[False,True],src=sv1pcr+sv1pcr,tgt=[],vol=17,srcdil=80.0/24)
sv2t7=trp.saveSamps(src=t72,tgt=[],vol=7,dil=4)
rt2=trp.runRT(pos=[True for i in t72+sv1t7]+[False for i in t72+sv1t7],src=t72+sv1t7+t72+sv1t7,tgt=[],vol=[12]+[9 for s in t72[1:]+sv1t7+t72+sv1t7],srcdil=2)
trp.diluteInPlace(tgt=rt2,dil=2)
lig2=trp.runLig(prefix=prodprefix,src=rt2+sv1rt,tgt=[],vol=[30]+[16 for s in rt2[1:]+sv1rt],srcdil=3)
qsamps=lig2+sv1t7+sv2t7 # Samples for QPCR
diltolig=[24 for s in t72]+[24*4 for s in sv1t7]+[24 for s in t72]+[24*4 for s in sv1t7]+[16 for s in sv1rt]+[8 for s in sv1t7]+[8 for s in sv2t7] # Their dilution so far from the T7 product point (before stop added)
dilneeded=[20000/d for d in diltolig]
qdil1=[max(50,math.sqrt(d)) for d in dilneeded] # Split dilution equally, but don't use more than 3ul in first step
qdil2=[dilneeded[i]/qdil1[i] for i in range(len(qdil1))] # Whatever remains
qpcrdil1=trp.runQPCRDIL(src=qsamps,tgt=[],vol=150,srcdil=qdil1) # First dilution before starting PCR (so the rest of the QPCR setup can be done while PCR is running)
pcr2=trp.runPCR(prefix=prodprefix,src="R1.T-.RT+.L"+prodprefix,tgt=[],vol=50,srcdil=4)
qpcrdil2x1=trp.runQPCRDIL(src=qpcrdil1,tgt=[],vol=150,srcdil=qdil2)
# qpcrdil2x2=trp.runQPCRDIL(src=qpcrdil1[0:2],tgt=['x2a','x2b'],vol=150,srcdil=[d*2 for d in qdil2[0:2]])
qpcrdil2x4=trp.runQPCRDIL(src=qpcrdil1[0:2],tgt=['x4a','x4b'],vol=150,srcdil=[d*4 for d in qdil2[0:2]])
qpcrdil2x4b=trp.runQPCRDIL(src=qpcrdil2x1[0:2],tgt=['x4ab','x4bb'],vol=150,srcdil=[4 for d in qdil2[0:2]])
qpcrdil2x16=trp.runQPCRDIL(src=qpcrdil2x1[0:2],tgt=['x16a','x16b'],vol=150,srcdil=[16 for d in qdil2[0:2]])
qpcrdil2=qpcrdil2x1+qpcrdil2x4+qpcrdil2x4b+qpcrdil2x16
trp.runQPCR(src=qpcrdil2,vol=15,srcdil=10.0/4)
trp.diluteInPlace(tgt=pcr2,dil=3)
sv2pcr=trp.saveSamps(src=pcr2,tgt=["R2"],vol=125,dil=1)
trp.finish()
tmpldil2 = trp.saveSamps(src=tmpldil1 + pcrsave,
vol=4,
dil=20,
plate=trp.e.DILPLATE)
tmpldil2b = trp.saveSamps(src=["BT371"],
vol=2,
dil=50,
plate=trp.e.DILPLATE)
tmpldil3 = trp.saveSamps(src=tmpldil2 + tmpldil2b,
vol=4,
dil=16,
dilutant=trp.r.SSD,
plate=trp.e.DILPLATE)
trp.runQPCR(src=ligtmp1 + ligtmp2,
vol=15,
srcdil=15.0 / 6,
primers=["A", "B"])
notSpiked = [t for t in tmpldil3 if "spike" not in t]
spiked = [t for t in tmpldil3 if "spike" in t]
print "Setting up qPCR of %d non-spiked template/PCR products with A,B,M,T primers" % (
len(notSpiked))
trp.runQPCR(src=notSpiked + ["Water"],
vol=15,
srcdil=15.0 / 6,
primers=["A", "B", "M", "T"])
print "Setting up qPCR of %d spiked template/PCR products with M,T primers" % (
len(tmpldil3[3:]))
trp.runQPCR(src=spiked, vol=15, srcdil=15.0 / 6, primers=["M", "T"])
trp.e.w.setOptimization(False)
trp.finish()
else:
reagents=Sample.getAllOnPlate(Experiment.REAGENTPLATE)+Sample.getAllOnPlate(Experiment.EPPENDORFS)
for r in reagents:
if r.volume<=0:
r.initvolume=-r.volume+r.plate.unusableVolume
Sample.clearall()
t71=trp.runT7Setup(theo=False,src=srcs*2,tgt=["%s.T%d"%(srcs[i],timepoints[j]) for j in range(len(timepoints)) for i in range(len(srcs))],vol=10,srcdil=10.0/6)
timesofar=0
for i in range(len(timepoints)):
lastelapse=trp.e.w.elapsed
trp.runT7Pgm(10,timepoints[i]-timesofar) # vol, duration
timesofar=timesofar+timepoints[i]
tp=trp.runT7Stop(theo=False,vol=10,tgt=t71[(i*len(srcs)):((i+1)*len(srcs))],stopmaster=stop)
print "Dead time=%.1f min"%((trp.e.w.elapsed-lastelapse)/60.0)
trp.diluteInPlace(tgt=t71,dil=5)
# Dilute input samples enough to use in qPCR directly (should be 5000/(rnagain*2*5) = 20)
qpcrdil1=trp.runQPCRDIL(src=t71,tgt=[],vol=100,srcdil=20,dilPlate=True)
rt1=trp.runRT(pos=True,src=t71,tgt=[],vol=10,srcdil=2)
rt1=trp.diluteInPlace(tgt=rt1,dil=5)
lig1=trp.runLig(src=rt1,tgt=[],vol=25,srcdil=3,master=ligmaster)
prods=trp.saveSamps(lig1,vol=5,dil=10,plate=Experiment.DILPLATE)
trp.runQPCR(src=qpcrdil1,vol=15,srcdil=10.0/4,primers=["WX","REF"])
trp.runQPCR(src=prods,vol=15,srcdil=10.0/4,primers=["WX","AX","REF"])
trp.finish()
for i in range(len(timepoints)):
lastelapse = trp.e.w.elapsed
trp.runT7Pgm(10, timepoints[i] - timesofar)
timesofar = timesofar + timepoints[i]
tp = trp.runT7Stop(theo=False,
vol=10,
tgt=t71[(i * len(srcs)):((i + 1) * len(srcs))],
stopmaster=stop)
print "Dead time=%.1f min" % ((trp.e.w.elapsed - lastelapse) / 60.0)
trp.diluteInPlace(tgt=t71, dil=5)
print t71
# Dilute input samples enough to use in qPCR directly (should be 5000/(rnagain*2*5) = 20)
qpcrdil1 = trp.runQPCRDIL(src=t71,
tgt=[],
vol=100,
srcdil=20,
dilPlate=True)
rt1 = trp.runRT(pos=True, src=t71, tgt=[], vol=5, srcdil=2)
rt1 = trp.diluteInPlace(tgt=rt1, dil=5)
lig1 = trp.runLig(src=rt1, tgt=[], vol=10, srcdil=3, master=ligmaster)
prods = trp.diluteInPlace(tgt=lig1, dil=10)
print "qpcrdil1=", qpcrdil1
trp.runQPCR(src=qpcrdil1, vol=15, srcdil=10.0 / 4, primers=tmplqpcr)
print "prods=", prods
trp.runQPCR(src=prods, vol=15, srcdil=10.0 / 4, primers=pcr)
trp.finish()
dilPlate=False)
rt1 = trp.runRT(pos=True, src=t71, tgt=[], vol=30, srcdil=2)
rt1 = trp.diluteInPlace(tgt=rt1, dil=5)
lig = trp.runLig(src=rt1 * 6,
tgt=[],
vol=20,
srcdil=3,
master=ligmaster1 * 3 + ligmaster2 * 3)
prods = trp.diluteInPlace(tgt=lig, dil=10)
print "prod=", prods
for i in range(len(qpcrdil1)):
trp.runQPCR(src=qpcrdil1[i],
vol=15,
srcdil=10.0 / 4,
primers=[tmplqpcr[i]],
nreplicates=3)
for p in pcr1:
trp.runQPCR(src=prods[0:3],
vol=15,
srcdil=10.0 / 4,
primers=p,
nreplicates=3)
for p in pcr2:
trp.runQPCR(src=prods[3:6],
vol=15,
srcdil=10.0 / 4,
primers=p,
nreplicates=3)
if r.volume<=0:
r.initvolume=-r.volume+r.plate.unusableVolume
Sample.clearall()
t71=trp.runT7Setup(theo=False,src=srcs*2,tgt=["%s.T%d"%(srcs[i],timepoints[j]) for j in range(len(timepoints)) for i in range(len(srcs))],vol=10,srcdil=10.0/6)
timesofar=0
for i in range(len(timepoints)):
lastelapse=trp.e.w.elapsed
trp.runT7Pgm(10,timepoints[i]-timesofar)
timesofar=timesofar+timepoints[i]
tp=trp.runT7Stop(theo=False,vol=10,tgt=t71[(i*len(srcs)):((i+1)*len(srcs))],stopmaster=stop)
print "Dead time=%.1f min"%((trp.e.w.elapsed-lastelapse)/60.0)
trp.diluteInPlace(tgt=t71,dil=5)
print t71
# Dilute input samples enough to use in qPCR directly (should be 5000/(rnagain*2*5) = 20)
qpcrdil1=trp.runQPCRDIL(src=t71,tgt=[],vol=100,srcdil=20,dilPlate=True)
rt1=trp.runRT(pos=True,src=t71,tgt=[],vol=5,srcdil=2)
rt1=trp.diluteInPlace(tgt=rt1,dil=5)
lig1=trp.runLig(src=rt1,tgt=[],vol=10,srcdil=3,master=ligmaster)
prods=trp.diluteInPlace(tgt=lig1,dil=10)
print "qpcrdil1=",qpcrdil1
trp.runQPCR(src=qpcrdil1,vol=15,srcdil=10.0/4,primers=tmplqpcr)
print "prods=",prods
trp.runQPCR(src=prods,vol=15,srcdil=10.0/4,primers=pcr)
trp.finish()
for i in range(len(timepoints)):
lastelapse = trp.e.w.elapsed
trp.runT7Pgm(10, timepoints[i] - timesofar) # vol, duration
timesofar = timesofar + timepoints[i]
tp = trp.runT7Stop(theo=False,
vol=10,
tgt=t71[(i * len(srcs)):((i + 1) * len(srcs))],
stopmaster=stop)
print "Dead time=%.1f min" % ((trp.e.w.elapsed - lastelapse) / 60.0)
trp.diluteInPlace(tgt=t71, dil=5)
# Dilute input samples enough to use in qPCR directly (should be 5000/(rnagain*2*5) = 20)
qpcrdil1 = trp.runQPCRDIL(src=t71,
tgt=[],
vol=100,
srcdil=20,
dilPlate=True)
rt1 = trp.runRT(pos=True, src=t71, tgt=[], vol=10, srcdil=2)
rt1 = trp.diluteInPlace(tgt=rt1, dil=5)
lig1 = trp.runLig(src=rt1, tgt=[], vol=25, srcdil=3, master=ligmaster)
prods = trp.saveSamps(lig1, vol=5, dil=10, plate=Experiment.DILPLATE)
trp.runQPCR(src=qpcrdil1, vol=15, srcdil=10.0 / 4, primers=["WX", "REF"])
trp.runQPCR(src=prods,
vol=15,
srcdil=10.0 / 4,
primers=["WX", "AX", "REF"])
trp.finish()
# But use PCR product from sample plate for next rount to avoid extra dilution
input=pcr2
if doqpcr:
# Setup qPCR
# Do A,B,M,T of input and PCR products (4*9), A,B of ligation products (2*8), M,T of reference
# Saved products should already be at correct concentration for qPCR
#trp.e.w.setOptimization(True)
print "Setting up qPCR of %d ligation products with A,B primers"%(len(ligsave1+ligsave2))
ligtmp1=trp.saveSamps(src=ligsave1,vol=4,dil=8,plate=trp.e.DILPLATE,dilutant=trp.r.SSD)
ligtmp2=trp.saveSamps(src=ligsave2,vol=4,dil=8*3.5,plate=trp.e.DILPLATE,dilutant=trp.r.SSD)
# Need to dilute templates to match PCR products
tmpldil1=trp.saveSamps(src=templates,vol=4,dil=20.0/3,plate=trp.e.DILPLATE)
tmpldil2=trp.saveSamps(src=tmpldil1+pcrsave,vol=4,dil=20,plate=trp.e.DILPLATE)
tmpldil2b=trp.saveSamps(src=["BT371"],vol=2,dil=50,plate=trp.e.DILPLATE)
tmpldil3=trp.saveSamps(src=tmpldil2+tmpldil2b,vol=4,dil=16,dilutant=trp.r.SSD,plate=trp.e.DILPLATE)
trp.runQPCR(src=ligtmp1+ligtmp2,vol=15,srcdil=15.0/6,primers=["A","B"])
notSpiked=[t for t in tmpldil3 if "spike" not in t]
spiked=[t for t in tmpldil3 if "spike" in t]
print "Setting up qPCR of %d non-spiked template/PCR products with A,B,M,T primers"%(len(notSpiked))
trp.runQPCR(src=notSpiked+["Water"],vol=15,srcdil=15.0/6,primers=["A","B","M","T"])
print "Setting up qPCR of %d spiked template/PCR products with M,T primers"%(len(tmpldil3[3:]))
trp.runQPCR(src=spiked,vol=15,srcdil=15.0/6,primers=["M","T"])
trp.e.w.setOptimization(False)
trp.finish()
