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)
for i in range(len(qpcrdil1)):
trp.runQPCR(src=qpcrdil1[i],vol=15,srcdil=10.0/4,primers=[tmplqpcr[i]])
Sample.clearall()
currprefix=srcprefix
if currprefix=='A':
altprefix='B'
else:
altprefix='A'
sv=[]
svligtype=[]
svdil=[]
t7all=[]
for round in range(ndblrounds):
# Round 1 (Keep uncleaved +theo)
t71=trp.runT7(theo=[True],src=input,vol=[10],srcdil=10.0/3,dur=15)
t71s=findsamps(t71)[0]
trp.e.waitpgm()
trp.e.w.userprompt("Check T7 volume in %s, should be %.1f ul"%(t71s.plate.wellname(t71s.well),t71s.volume))
t7all=t7all+t71
rt1=trp.runRT(pos=[True],src=t71,vol=[10],srcdil=2)
rt1s=findsamps(rt1)[0]
trp.e.waitpgm()
trp.e.w.userprompt("Check RT volume in %s, should be %.1f ul"%(rt1s.plate.wellname(rt1s.well),rt1s.volume))
trp.diluteInPlace(tgt=t71,dil=5) # Dilute more to conserve
trp.diluteInPlace(tgt=rt1,dil=3)
if doqpcr:
sv=sv+trp.saveSamps(src=rt1,vol=8,dil=5)
svligtype=svligtype+[altprefix]
svdil=svdil+[2*2*3*5]
pcr1=trp.runPCR(prefix=[currprefix],src=rt1,vol=25,srcdil=4,ncycles=cycles1)
pcr1s=findsamps(pcr1)[0]
