else:
reagents = Sample.getAllOnPlate(Experiment.REAGENTPLATE)
for r in reagents:
if r.volume < 0:
r.initvolume = -r.volume + 20
Sample.clearall()
# Round 1 (Keep uncleaved +theo)
t71 = trp.runT7(theo=[False, True],
src=[input, input],
tgt=[],
vol=[16, 18],
srcdil=4)
sv1t7 = trp.saveSamps(src=t71, tgt=[], vol=10, dil=[4, 4])
rt1 = trp.runRT(pos=[True, True], src=t71, tgt=[], vol=[15, 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=4)
sv2t7 = trp.saveSamps(src=t72, tgt=[], vol=7, dil=[4, 4])
rt2 = trp.runRT(pos=[True
for i in t72 + sv1t7] + [False for i in t72 + sv1t7],
src=t72 + sv1t7 + t72 + sv1t7,
else:
reagents = Sample.getAllOnPlate(Experiment.REAGENTPLATE)
for r in reagents:
if r.volume < 0:
r.initvolume = -r.volume + 20
Sample.clearall()
# Round 1
t71 = trp.runT7(theo=[False, True],
src=["IN", "IN"],
tgt=[],
vol=[10, 20],
srcdil=10.0 / 3)
sv1 = trp.saveSamps(src=t71, tgt=[], vol=[10, 10], dil=[4, 4])
rt1 = trp.runRT(pos=[True], src=["IN.T+"], tgt=[], vol=30, srcdil=2)
trp.diluteInPlace(tgt=rt1, dil=2)
sv2 = trp.saveSamps(src=rt1, tgt=[], vol=20, dil=2)
pcr1 = trp.runPCR(prefix=["A"], src=rt1, tgt=[], vol=100, srcdil=4)
sv3 = trp.saveSamps(src=pcr1, tgt=["R1"], vol=85, dil=3)
# Round 2
t72 = trp.runT7(theo=[False, True],
src=sv3 + sv3,
tgt=[],
vol=[15, 10],
srcdil=10.0 / 3)
rt2 = trp.runRT(pos=[True for i in t72 + sv1] + [False for i in t72 + sv1],
src=t72 + sv1 + t72 + sv1,
tgt=[],
vol=[30, 8, 8, 8, 8, 8, 8, 8],
srcdil=2)
trp=TRP()
if iteration==0:
trp.addTemplates([input],200) # Add a template with stock concentration 200nM
else:
reagents=Sample.getAllOnPlate(Experiment.REAGENTPLATE)
for r in reagents:
if r.volume<0:
r.initvolume=-r.volume+20
Sample.clearall()
# Round 1 (Keep uncleaved +theo)
t71=trp.runT7(theo=[False,True],src=[input,input],tgt=[],vol=[16,18],srcdil=4)
sv1t7=trp.saveSamps(src=t71,tgt=[],vol=10,dil=[4,4])
rt1=trp.runRT(pos=[True,True],src=t71,tgt=[],vol=[15,22],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=4)
sv2t7=trp.saveSamps(src=t72,tgt=[],vol=7,dil=[4,4])
rt2=trp.runRT(pos=[True for i in t72+sv1t7]+[False for i in t72+sv1t7],src=t72+sv1t7+t72+sv1t7,tgt=[],vol=[11,9,9,9,9,9,9,9],srcdil=2)
trp.diluteInPlace(tgt=rt2,dil=2)
lig2=trp.runLig(prefix=prodprefix,src=rt2+sv1rt,tgt=[],vol=[30,15,15,15,15,15,15,15,15,15],srcdil=3)
qsamps=lig2+sv1t7+sv2t7 # Samples for QPCR
diltolig=[24,24,24*4,24*4,24,24,24*4,24*4,24*2,24*2]+[8,8]+[8,8] # 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
trp=TRP()
if iteration==0:
trp.addTemplates(["IN"],200) # Add a template with stock concentration 200nM
else:
reagents=Sample.getAllOnPlate(Experiment.REAGENTPLATE)
for r in reagents:
if r.volume<0:
r.initvolume=-r.volume+20
Sample.clearall()
# Round 1
t71=trp.runT7(theo=[False,True],src=["IN","IN"],tgt=[],vol=[10,20],srcdil=10.0/3)
sv1=trp.saveSamps(src=t71,tgt=[],vol=[10,10],dil=[4,4])
rt1=trp.runRT(pos=[True],src=["IN.T+"],tgt=[],vol=30,srcdil=2)
trp.diluteInPlace(tgt=rt1,dil=2)
sv2=trp.saveSamps(src=rt1,tgt=[],vol=20,dil=2)
pcr1=trp.runPCR(prefix=["A"],src=rt1,tgt=[],vol=100,srcdil=4)
sv3=trp.saveSamps(src=pcr1,tgt=["R1"],vol=85,dil=3)
# Round 2
t72=trp.runT7(theo=[False,True],src=sv3+sv3,tgt=[],vol=[15,10],srcdil=10.0/3)
rt2=trp.runRT(pos=[True for i in t72+sv1]+[False for i in t72+sv1],src=t72+sv1+t72+sv1,tgt=[],vol=[30,8,8,8,8,8,8,8],srcdil=2)
trp.diluteInPlace(tgt=rt2,dil=2)
lig2=trp.runLig(prefix="B",src=rt2+sv2,tgt=[],vol=[69,12,12,12,12,12,12,12,12],srcdil=3.0)
dilneeded=6000/12
qpcrdil1=trp.runQPCRDIL(src=lig2,vol=100,srcdil=math.sqrt(dilneeded))
dilneeded=math.sqrt(dilneeded)
pcr2=trp.runPCR(prefix="B",src="R1.T-.RT+.LB",tgt=["R2.1","R2.2"],vol=100,srcdil=4)
qpcrdil2=trp.runQPCRDIL(src=qpcrdil1,vol=100,srcdil=math.sqrt(6000.0/12))
trp.runQPCR(src=qpcrdil2,vol=10,srcdil=10.0/4)