예제 #1
0

for iteration in range(2):
    print "Iteration ",iteration+1
    trp=TRP()

    if iteration==0:
        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:
예제 #2
0
                         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=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)
예제 #3
0
for iteration in range(2):
    print "Iteration ",iteration+1
    trp=TRP()
    if iteration==0:
        trp.addTemplates([input,ctl],160)   # Add a template with stock concentration 80nM
    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,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
예제 #4
0
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=[],
예제 #5
0
                r.initvolume = -r.volume + 20
        Sample.clearall()

    currprefix = srcprefix
    if currprefix == 'A':
        altprefix = 'B'
    else:
        altprefix = 'A'
    ligsave1 = []
    ligsave2 = []
    pcrsave = []
    input = templates

    for round in range(ndblrounds):
        # Round 1 (Keep uncleaved +theo)
        t71 = trp.runT7(theo=True, src=input, vol=12, srcdil=10.0 / 3, dur=15)
        rt1 = trp.runRT(pos=True, src=t71, vol=10, srcdil=2)
        t71 = trp.diluteInPlace(tgt=t71, dil=5)  # Dilute more to conserve
        rt1 = trp.diluteInPlace(tgt=rt1, dil=3.5)

        # Save RT product so can do ligation during 2nd round
        sv1rt = trp.saveSamps(src=rt1, vol=8, dil=3, plate=trp.e.DILPLATE)

        prodbase = "R%d-%c" % (firstround + round * 2, currprefix)
        pcr1 = trp.runPCR(prefix=currprefix,
                          src=rt1,
                          tgt=[prodbase, prodbase + "-spike"],
                          vol=pcrvol,
                          srcdil=4,
                          ncycles=cycles1)
        pcr1 = trp.diluteInPlace(tgt=pcr1, dil=2)
예제 #6
0
                r.initvolume=-r.volume+20
        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)
예제 #7
0
    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=12,
                        srcdil=10.0 / 3,
                        dur=15)
        t7all = t7all + t71
        rt1 = trp.runRT(pos=[True], src=t71, vol=[10], srcdil=2)
        trp.diluteInPlace(tgt=t71, dil=5)  # Dilute more to conserve
        trp.diluteInPlace(
            tgt=rt1, dil=9
        )  # Dilute same as combined RT+Ligation steps in second half of double-round, reduce inhibition of PCR
        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,
예제 #8
0
reagents=None

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()
예제 #9
0
                r.initvolume=-r.volume+20
        Sample.clearall()

    currprefix=srcprefix
    if currprefix=='A':
        altprefix='B'
    else:
        altprefix='A'
    ligsave1=[]
    ligsave2=[]
    pcrsave=[]
    input=templates
    
    for round in range(ndblrounds):
        # Round 1 (Keep uncleaved +theo)
        t71=trp.runT7(theo=True,src=input,vol=12,srcdil=10.0/3,dur=15)
        rt1=trp.runRT(pos=True,src=t71,vol=10,srcdil=2)
        t71=trp.diluteInPlace(tgt=t71,dil=5)  # Dilute more to conserve
        rt1=trp.diluteInPlace(tgt=rt1,dil=3.5)

        # Save RT product so can do ligation during 2nd round
        sv1rt=trp.saveSamps(src=rt1,vol=8,dil=3,plate=trp.e.DILPLATE)

        prodbase="R%d-%c"%(firstround+round*2,currprefix)
        pcr1=trp.runPCR(prefix=currprefix,src=rt1,tgt=[prodbase,prodbase+"-spike"],vol=pcrvol,srcdil=4,ncycles=cycles1)
        pcr1=trp.diluteInPlace(tgt=pcr1,dil=2)
        eppie=trp.saveSamps(src=pcr1,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) (also eppie may be removed before end)
        pcrsave=pcrsave+trp.saveSamps(src=eppie,vol=5,dil=20, plate=trp.e.DILPLATE)

        # Round 2 (-theo, Ligate, keep cleaved)
예제 #10
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                r.initvolume=-r.volume+20
        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=12,srcdil=10.0/3,dur=15)
        t7all=t7all+t71
        rt1=trp.runRT(pos=[True],src=t71,vol=[10],srcdil=2)
        trp.diluteInPlace(tgt=t71,dil=5)  # Dilute more to conserve
        trp.diluteInPlace(tgt=rt1,dil=9)   # Dilute same as combined RT+Ligation steps in second half of double-round, reduce inhibition of PCR
        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)
        trp.diluteInPlace(tgt=pcr1,dil=6)
        sv1pcr=trp.saveSamps(src=pcr1,tgt=["R%d-%c"%(firstround+round*2,currprefix)],vol=125,dil=1,plate=trp.e.EPPENDORFS)
    
        # Round 2 (-theo, Ligate, keep cleaved)
        t72=trp.runT7(theo=[False],src=sv1pcr,vol=12,srcdil=10.0/3)
        t7all=t7all+t72