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()
t71master=["%s.MT"%s for s in srcs]
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)
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)
if iteration==0:
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.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"])
r.initvolume=-r.volume+r.plate.unusableVolume
Sample.clearall()
t71master=["%s.MT"%s for s in srcs]
trp.runT7Setup(theo=False,src=srcs,tgt=t71master,vol=len(timepoints)*(t7vol*1.02+2)+1+15+20,srcdil=10.0/6)
startTime=trp.e.w.elapsed
t71=[]
stopDelay=0*60
for i in range(len(timepoints)):
trp.e.sanitize()
pauseTime=(timepoints[i]*60-(trp.e.w.elapsed-startTime)-stopDelay)
if pauseTime>0:
print "Pausing for %.1f minutes"%(pauseTime/60.0)
trp.e.w.userprompt("Pausing to incubate first T7 at room temperature...",pauseTime)
tp=trp.saveSamps(src=t71master,tgt=["%s.T%d"%(s,timepoints[i]) for s in srcs],vol=t7vol,dil=1,plate=trp.e.SAMPLEPLATE)
trp.runT7Stop(theo=False,vol=t7vol,tgt=tp,stopmaster=stop)
print "T7 stop %d done at %.1f minutes"%(i,(trp.e.w.elapsed-startTime)/60.0)
trp.diluteInPlace(tgt=tp,dil=5)
trp.e.w.flushQueue()
t71=t71+tp
# 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=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]])
