# Round 1 (Keep uncleaved +theo) t71=trp.runT7(theo=[False,True,False],src=[input,input,ctl],tgt=[],vol=18,srcdil=80.0/24) sv1t7=trp.saveSamps(src=t71,tgt=[],vol=10,dil=4) rt1=trp.runRT(pos=[True,True,True ],src=t71,tgt=[],vol=22,srcdil=2) trp.diluteInPlace(tgt=rt1,dil=2) sv1rt=trp.saveSamps(src=rt1,tgt=[],vol=15,dil=2) 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)
# 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 for s in rt2] + [prodprefix, prodprefix, ctlprodprefix],
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 * 3 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 = [10000 / d for d in diltolig]
qdil1 = [40 for d in dilneeded] # 40x for first dilution
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()
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)
# Round 2 (Keep uncleaved +theo)
print "***** Round 2 T7 *****"
in2=pcr1+["Neg"]
t72=trp.runT7New(theo=[True for i in in2]+[False for i in in2],src=in2+in2,tgt=["%s.T2+"%i for i in in2]+["%s.T2-"%i for i in in2],vol=10,srcdil=10.0/3)
sv2t7=trp.saveSamps(src=t72,vol=3,dil=25,plate=trp.e.DILPLATE)
t72 = trp.runT7(theo=False, src=pcr1, vol=12, srcdil=10.0 / 3)
rt2 = trp.runRT(pos=True, src=t72, vol=10, srcdil=2)
t72 = trp.diluteInPlace(tgt=t72, dil=5) # Dilute more to conserve
rt2 = trp.diluteInPlace(tgt=rt2, dil=3)
# Swap prefixes
altprefix = currprefix
if currprefix == "B":
currprefix = "A"
else:
currprefix = "B"
# Run ligation of the first-half round too
lig2 = trp.runLig(prefix=currprefix,
src=sv1rt + rt2,
vol=[17, 17, 25, 25],
srcdil=3)
lig2 = trp.diluteInPlace(tgt=lig2, dil=1.33)
# Save ligation products for qPCR (note that round 1 had more dilution of RT product, so back that out here)
ligsave1 = ligsave1 + trp.saveSamps(src=lig2[:2],
vol=5,
dil=20,
plate=trp.e.DILPLATE,
dilutant=trp.r.SSD)
ligsave2 = ligsave2 + trp.saveSamps(src=lig2[2:],
vol=5,
dil=20,
plate=trp.e.DILPLATE,
dilutant=trp.r.SSD)
prodbase = "R%d-%c" % (firstround + 1 + round * 2, currprefix)
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
t72 = trp.runT7New(
theo=[False for i in r2in] + [True for i in r2in], src=r2in + r2in, tgt=[], vol=10, srcdil=10.0 / 3
)
trp.diluteInPlace(tgt=t72, dil=5)
sv2t7 = trp.saveSamps(src=t72, vol=10, dil=10, plate=trp.e.DILPLATE)
rt2 = trp.runRT(pos=True, src=t72, tgt=[], vol=5, srcdil=2)
trp.diluteInPlace(tgt=rt2, dil=4)
lig2 = trp.runLig(prefix=prodprefix, src=rt2, tgt=[], vol=10, srcdil=3)
trp.diluteInPlace(tgt=lig2, dil=5)
qsamps = lig2 + sv2t7 # Samples for QPCR
diltolig = [2 * 5 * 2 * 4 * 3 * 5 for d in lig2] + [
2 * 5 * 10 * rnagain for d in sv2t7
] # Their dilution so far from the T7 product point (before stop added)
dilneeded = [10000.0 / d for d in diltolig]
# qdil1=[40 for d in dilneeded] # 40x for first dilution
# qdil2=[dilneeded[i]/qdil1[i] for i in range(len(qdil1))] # Whatever remains
# qpcrdil1=trp.runQPCRDIL(src=qsamps,tgt=[],vol=100,srcdil=qdil1,dilPlate=False) # First dilution before starting PCR (so the rest of the QPCR setup can be done while PCR is running)
ligconc = (endconc / 3.0) * 3.0 / 10.0 * rnagain / diltolig[0] # Expected concentration of ligation product here
cycles = math.ceil(math.log(endconc / ligconc, pcreff))
# Amplify to end of exponential phase
print "PCR input conc=%.3g pM, PCR cycles=%.1f, End Conc=%.0f nM" % (ligconc * 1e12, cycles, endconc * 1e9)
pcr2 = trp.runPCR(prefix=prodprefix, src=lig2[0 : len(sv1pcr)], tgt=[], vol=25, srcdil=4, ncycles=cycles)
qpcrdil2 = trp.runQPCRDIL(src=qsamps, tgt=[], vol=100, srcdil=dilneeded, dilPlate=True)
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,
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)
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],
src=r2in + r2in,
tgt=[],
vol=10,
srcdil=10.0 / 3)
trp.diluteInPlace(tgt=t72, dil=5)
sv2t7 = trp.saveSamps(src=t72, vol=10, dil=10, plate=trp.e.DILPLATE)
rt2 = trp.runRT(pos=True, src=t72, tgt=[], vol=5, srcdil=2)
trp.diluteInPlace(tgt=rt2, dil=4)
lig2 = trp.runLig(prefix=prodprefix, src=rt2, tgt=[], vol=10, srcdil=3)
trp.diluteInPlace(tgt=lig2, dil=5)
qsamps = lig2 + sv2t7 # Samples for QPCR
diltolig = [2 * 5 * 2 * 4 * 3 * 5 for d in lig2] + [
2 * 5 * 10 * rnagain for d in sv2t7
] # Their dilution so far from the T7 product point (before stop added)
dilneeded = [10000.0 / d for d in diltolig]
# qdil1=[40 for d in dilneeded] # 40x for first dilution
# qdil2=[dilneeded[i]/qdil1[i] for i in range(len(qdil1))] # Whatever remains
# qpcrdil1=trp.runQPCRDIL(src=qsamps,tgt=[],vol=100,srcdil=qdil1,dilPlate=False) # First dilution before starting PCR (so the rest of the QPCR setup can be done while PCR is running)
ligconc = (endconc / 3.0) * 3.0 / 10.0 * rnagain / diltolig[
0] # Expected concentration of ligation product here
cycles = math.ceil(math.log(endconc / ligconc, pcreff))
# Amplify to end of exponential phase
print "PCR input conc=%.3g pM, PCR cycles=%.1f, End Conc=%.0f nM" % (
ligconc * 1e12, cycles, endconc * 1e9)
for iteration in range(2):
print "Iteration ",iteration+1
trp=TRP()
if iteration==0:
trp.addTemplates(input,8) # Add a template with stock concentration 8nM
else:
reagents=Sample.getAllOnPlate(Experiment.REAGENTPLATE)
for r in reagents:
if r.volume<0:
r.initvolume=-r.volume+20
Sample.clearall()
t71=trp.runT7(theo=[False for i in inputs]+[True for i in inputs],src=inputs+inputs,tgt=[],vol=10,srcdil=80.0/24,dur=15)
trp.diluteInPlace(tgt=t71,dil=2)
rt1=trp.runRT(pos=True,src=t71,tgt=[],vol=5,srcdil=2)
trp.diluteInPlace(tgt=rt1,dil=4)
lig1=trp.runLig(prefix=[prodprefixes[i%len(prodprefixes)] for i in range(len(rt1))],src=rt1,tgt=[],vol=10,srcdil=3)
trp.diluteInPlace(tgt=lig1,dil=4)
# Dilute input samples
qpcrdil1=trp.runQPCRDIL(src=inputs,tgt=[],vol=100,srcdil=40,dilPlate=True)
diltohere=[6*40 for s in inputs]+[2*2*2*4*3*4 for s in lig1] # Their dilution so far from the T7 product point (before stop added)
dilneeded=[10000/d for d in diltohere]
# trp.e.w.userprompt("Load QPCR plate and press return to start QPCR setup")
qpcrdil2=trp.runQPCRDIL(src=qpcrdil1+lig1,tgt=[],vol=100,srcdil=dilneeded,dilPlate=True)
trp.runQPCR(src=qpcrdil2,vol=15,srcdil=10.0/4)
trp.finish()
rt2 = trp.runRT(pos=True, src=t72, vol=[10], srcdil=2)
trp.diluteInPlace(tgt=t72, dil=5) # Dilute more to conserve
trp.diluteInPlace(tgt=rt2, dil=3)
if doqpcr:
sv = sv + trp.saveSamps(src=rt2, vol=8, dil=5)
svligtype = svligtype + [altprefix]
svdil = svdil + [2 * 2 * 3 * 5]
altprefix = currprefix
if currprefix == "B":
currprefix = "A"
else:
currprefix = "B"
if round == ndblrounds - 1 and doqpcr:
# Do the analysis as well
lig2 = trp.runLig(prefix=[currprefix] + svligtype,
src=rt2 + sv,
vol=[19] + [10 for s in sv],
srcdil=3)
qsamps = lig2[1:] # Samples for QPCR
lig2 = lig2[0:1]
trp.diluteInPlace(tgt=qsamps, dil=5)
qpcrdilt7 = trp.runQPCRDIL(
src=t7all, tgt=[], vol=100, srcdil=33.33,
dilPlate=True) # Dilute T7 products first
qsamps = qsamps + qpcrdilt7
dilneeded = [10000.0 / (d * 3 * 5) for d in svdil
] + [10000.0 / (2 * 5 * 33.33) for d in qpcrdilt7]
qpcrdil1 = trp.runQPCRDIL(
src=qsamps, tgt=[], vol=100, srcdil=dilneeded, dilPlate=True
) # First dilution before starting PCR (so the rest of the QPCR setup can be done while PCR is running)
else:
lig2 = trp.runLig(prefix=currprefix, src=rt2, vol=[24], srcdil=3)
if r.volume < 0:
r.initvolume = -r.volume + 20
Sample.clearall()
t71 = trp.runT7(theo=[False for i in inputs] + [True for i in inputs],
src=inputs + inputs,
tgt=[],
vol=10,
srcdil=80.0 / 24,
dur=15)
trp.diluteInPlace(tgt=t71, dil=2)
rt1 = trp.runRT(pos=True, src=t71, tgt=[], vol=5, srcdil=2)
trp.diluteInPlace(tgt=rt1, dil=4)
lig1 = trp.runLig(prefix=prodprefixes + prodprefixes,
src=rt1,
tgt=[],
vol=10,
srcdil=3)
trp.diluteInPlace(tgt=lig1, dil=4)
# Dilute input samples
qpcrdil1 = trp.runQPCRDIL(src=inputs,
tgt=[],
vol=100,
srcdil=40,
dilPlate=True)
diltohere = [6 * 40 for s in inputs] + [
2 * 2 * 2 * 4 * 3 * 4 for s in lig1
] # Their dilution so far from the T7 product point (before stop added)
dilneeded = [10000 / d for d in diltohere]
# trp.e.w.userprompt("Load QPCR plate and press return to start QPCR setup")
qpcrdil2 = trp.runQPCRDIL(src=qpcrdil1 + lig1,
# Round 2 (-theo, Ligate, keep cleaved)
t72=trp.runT7(theo=False,src=pcr1,vol=12,srcdil=10.0/3)
rt2=trp.runRT(pos=True,src=t72,vol=10,srcdil=2)
t72=trp.diluteInPlace(tgt=t72,dil=5) # Dilute more to conserve
rt2=trp.diluteInPlace(tgt=rt2,dil=3)
# Swap prefixes
altprefix=currprefix
if currprefix=="B":
currprefix="A"
else:
currprefix="B"
# Run ligation of the first-half round too
lig2=trp.runLig(prefix=currprefix,src=sv1rt+rt2,vol=[17,17,25,25],srcdil=3)
lig2=trp.diluteInPlace(tgt=lig2,dil=1.33)
# Save ligation products for qPCR (note that round 1 had more dilution of RT product, so back that out here)
ligsave1=ligsave1+trp.saveSamps(src=lig2[:2],vol=5,dil=20,plate=trp.e.DILPLATE,dilutant=trp.r.SSD)
ligsave2=ligsave2+trp.saveSamps(src=lig2[2:],vol=5,dil=20,plate=trp.e.DILPLATE,dilutant=trp.r.SSD)
prodbase="R%d-%c"%(firstround+1+round*2,currprefix)
pcr2=trp.runPCR(prefix=currprefix,tgt=[prodbase,prodbase+"-spike"],src=lig2[2:4],vol=pcrvol,srcdil=4,ncycles=cycles2)
pcr2=trp.diluteInPlace(tgt=pcr2,dil=2)
# Save PCR product in eppendorfs for archive
eppie= trp.saveSamps(src=pcr2,tgt=[prodbase+".D3",prodbase+"-spike.D3"],vol=26,dil=3,plate=trp.e.EPPENDORFS)
# And save in dilution plate for qPCR (will need 400x dilution from this point)
pcrsave=pcrsave+trp.saveSamps(src=eppie,vol=5,dil=20, plate=trp.e.DILPLATE)
for iteration in range(2):
print "Iteration ",iteration+1
trp=TRP()
if iteration==0:
trp.addTemplates(input,8) # Add a template with stock concentration 8nM
else:
reagents=Sample.getAllOnPlate(Experiment.REAGENTPLATE)
for r in reagents:
if r.volume<0:
r.initvolume=-r.volume+20
Sample.clearall()
t71=trp.runT7(theo=[False for i in inputs]+[True for i in inputs],src=inputs+inputs,tgt=[],vol=10,srcdil=80.0/24,dur=15)
trp.diluteInPlace(tgt=t71,dil=2)
rt1=trp.runRT(pos=True,src=t71,tgt=[],vol=5,srcdil=2)
trp.diluteInPlace(tgt=rt1,dil=4)
lig1=trp.runLig(prefix=prodprefixes+prodprefixes,src=rt1,tgt=[],vol=10,srcdil=3)
trp.diluteInPlace(tgt=lig1,dil=4)
# Dilute input samples
qpcrdil1=trp.runQPCRDIL(src=inputs,tgt=[],vol=100,srcdil=40,dilPlate=True)
diltohere=[6*40 for s in inputs]+[2*2*2*4*3*4 for s in lig1] # Their dilution so far from the T7 product point (before stop added)
dilneeded=[10000/d for d in diltohere]
# trp.e.w.userprompt("Load QPCR plate and press return to start QPCR setup")
qpcrdil2=trp.runQPCRDIL(src=qpcrdil1+lig1,tgt=[],vol=100,srcdil=dilneeded,dilPlate=True)
trp.runQPCR(src=qpcrdil2,vol=15,srcdil=10.0/4)
trp.finish()
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()
if r.volume < 0:
r.initvolume = -r.volume + 20
Sample.clearall()
t71 = trp.runT7(theo=[False for i in inputs] + [True for i in inputs],
src=inputs + inputs,
tgt=[],
vol=10,
srcdil=80.0 / 24,
dur=15)
trp.diluteInPlace(tgt=t71, dil=2)
rt1 = trp.runRT(pos=True, src=t71, tgt=[], vol=5, srcdil=2)
trp.diluteInPlace(tgt=rt1, dil=4)
lig1 = trp.runLig(
prefix=[prodprefixes[i % len(prodprefixes)] for i in range(len(rt1))],
src=rt1,
tgt=[],
vol=10,
srcdil=3)
trp.diluteInPlace(tgt=lig1, dil=4)
# Dilute input samples
qpcrdil1 = trp.runQPCRDIL(src=inputs,
tgt=[],
vol=100,
srcdil=40,
dilPlate=True)
diltohere = [6 * 40 for s in inputs] + [
2 * 2 * 2 * 4 * 3 * 4 for s in lig1
] # Their dilution so far from the T7 product point (before stop added)
dilneeded = [10000 / d for d in diltohere]
# trp.e.w.userprompt("Load QPCR plate and press return to start QPCR setup")
qpcrdil2 = trp.runQPCRDIL(src=qpcrdil1 + lig1,
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()
t7all=t7all+t72
rt2=trp.runRT(pos=True,src=t72,vol=[10],srcdil=2)
trp.diluteInPlace(tgt=t72,dil=5) # Dilute more to conserve
trp.diluteInPlace(tgt=rt2,dil=3)
if doqpcr:
sv=sv+trp.saveSamps(src=rt2,vol=8,dil=5)
svligtype=svligtype+[altprefix]
svdil=svdil+[2*2*3*5]
altprefix=currprefix
if currprefix=="B":
currprefix="A"
else:
currprefix="B"
if round==ndblrounds-1 and doqpcr:
# Do the analysis as well
lig2=trp.runLig(prefix=[currprefix]+svligtype,src=rt2+sv,vol=[19]+[10 for s in sv],srcdil=3)
qsamps=lig2[1:] # Samples for QPCR
lig2=lig2[0:1]
trp.diluteInPlace(tgt=qsamps,dil=5)
qpcrdilt7=trp.runQPCRDIL(src=t7all,tgt=[],vol=100,srcdil=33.33,dilPlate=True) # Dilute T7 products first
qsamps=qsamps+qpcrdilt7
dilneeded=[10000.0/(d*3*5) for d in svdil]+[10000.0/(2*5*33.33) for d in qpcrdilt7]
qpcrdil1=trp.runQPCRDIL(src=qsamps,tgt=[],vol=100,srcdil=dilneeded,dilPlate=True) # First dilution before starting PCR (so the rest of the QPCR setup can be done while PCR is running)
else:
lig2=trp.runLig(prefix=currprefix,src=rt2,vol=[24],srcdil=3)
pcr2=trp.runPCR(prefix=currprefix,src=lig2,vol=25,srcdil=4,ncycles=cycles2)
if round==ndblrounds-1 and doqpcr:
trp.e.w.userprompt("Press return to start QPCR setup")
trp.runQPCR(src=qpcrdil1,vol=15,srcdil=10.0/4)
trp.diluteInPlace(tgt=pcr2,dil=6)
input=trp.saveSamps(src=pcr2,tgt=["R%d-%c"%(firstround+1+round*2,currprefix)],vol=125,dil=1,plate=trp.e.EPPENDORFS)