Beispiel #1
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
    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)
Beispiel #2
0
    # 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)

    rt2=trp.runRT(pos=True,src=t72,tgt=[],vol=5,srcdil=2)
    trp.diluteInPlace(tgt=rt2,dil=4)

    lig2=trp.runLig(prefix="A",src=rt2,tgt=[],vol=10,srcdil=3)
    trp.diluteInPlace(tgt=lig2,dil=5)
    sv2lig=trp.saveSamps(src=lig2,vol=20,dil=5,plate=trp.e.DILPLATE)
Beispiel #3
0
    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)
        t72 = trp.runT7(theo=False, src=pcr1, vol=12, srcdil=10.0 / 3)
Beispiel #4
0
     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]
     trp.e.waitpgm()
     trp.e.w.userprompt("Check PCR volume in %s, should be %.1f ul"%(pcr1s.plate.wellname(pcr1s.well),pcr1s.volume))
     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=10,srcdil=10.0/3)
     # 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]
Beispiel #5
0
        for r in reagents:
            if r.volume < 0:
                r.initvolume = -r.volume + 20
        Sample.clearall()

    # Round 1 (Keep uncleaved +theo)
    t71 = trp.runT7New(theo=True,
                       src=inputs,
                       tgt=["%s.T1+" % i for i in inputs],
                       vol=10,
                       srcdil=10.0 / 3,
                       dur=30)
    trp.diluteInPlace(tgt=t71, dil=5)
    rt1 = trp.runRT(pos=True, src=t71, tgt=[], vol=5, srcdil=2)
    trp.diluteInPlace(tgt=rt1, dil=4)
    pcr1 = trp.runPCR(prefix=srcprefix, src=rt1, tgt=[], vol=25, srcdil=4)
    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)
Beispiel #6
0
                       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)
Beispiel #7
0
                        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
        rt2 = trp.runRT(pos=True, src=t72, vol=[10], srcdil=2)
        trp.diluteInPlace(tgt=t72, dil=5)  # Dilute more to conserve
Beispiel #8
0
    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)
        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":
Beispiel #9
0
            if r.volume < 0:
                r.initvolume = -r.volume + 20
        Sample.clearall()

    # Round 1 (Keep uncleaved +theo)
    t71 = trp.runT7New(theo=True, src=inputs, tgt=["%s.T1+" % i for i in inputs], vol=10, srcdil=10.0 / 3, dur=30)
    trp.diluteInPlace(tgt=t71, dil=5)
    rt1 = trp.runRT(pos=True, src=t71, tgt=[], vol=5, srcdil=2)
    trp.diluteInPlace(tgt=rt1, dil=4)

    rtconc = tmplConc * 3.0 / 10.0 * rnagain / (2 * 5 * 2 * 4 * 4)  # Expected concentration of ligation product here
    cycles = math.ceil(math.log(endconc / rtconc, pcreff))
    # Amplify to end of exponential phase
    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
Beispiel #10
0
 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
     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":