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]