Summary of the modifications for automated rep-PCRa

Rep-PCR conditionMethod
    Reaction vol (μl)5025
    Template (ng)10050
    Primer(s)References 19 and 44References 19 and 44
    BufferGitschierb plus DMSO and BSAcABI; no DMSO or BSAc
    Annealing temp (°C)38-6050-70
    Cycling parameters95°C for 7 min, 35 cycles (each cycle, 90°C for 30 s, annealing for 1 min, and 65°C for 8 min), 65°C for 16 min, and 4°C holding94°C for 2 min, 35 cycles (each cycle, 94°C for 30 s, annealing for 30 s, and 70°C for 1.5 min), 70°C for 3 min, and 4°C holding
    Cycle time (h)6.52
    Fragment size50 bp-10 kb50 bp-4 kb
Amplicon separation and detectionAgarose gelMicrofluidics
    Assay setup1 h5 min
    Total run time6 h55 min
    Biohazardous wasteEthidium bromide, UV light exposure<30 μl of 1.5% acrylamide in chip
    Amplicon vol (μl)101
    Data captureHigh-resolution digital camera; manuallyAgilent 2100 bioanalyzer; automatically
    Time for 12 samples (h)81
Analysis and reportingBioNumericsDiversiLab
    Stable algorithmNoYes
    InterfaceLocal stand-alone or networkSecure, internet based; unlimited access
    Data uploadManualAutomatic
    Sample demographicsRequired for each gelSingle entry for each isolate
    ReportNoMultiple options
  • a This table is intended for general comparison of manual and automated rep-PCR protocols; some conditions may vary depending on source.

  • b Reference 44.

  • c DMSO, dimethyl sulfoxide; BSA, bovine serum albumin; ABI, Applied Biosystems, Inc.