# 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)