Table 2: The protocol of the long-range PCR reactions for different enzymes

From: Long-range PCR in next-generation sequencing: comparison of six enzymes and evaluation on the MiSeq sequencer

EnzymesThe composition of reaction mixturePCR conditions
SequalPrep70 ng template DNA, 2 μL of a 2.5 μmol/L primers, 2 μL 10× Reaction Buffer, 1 μL PCR enhancer, 0.4 μL DMSO, 0.36 μL Polymerase and sterilized distilled water up to 20 μL94°C 2 minutes
  10 cycles
    94° 10 seconds
    Tm-5° (55, 60 or 65°C) 30 seconds
    68°C 10/13 minutes
  25 cycles
    94°C 10 seconds
   Tm-5° (55, 60 or 65°C) 30 seconds
   68°C 10/13 minutes plus 20 seconds
  72°C 5 minutes
  hold at 4°C
AccuPrime70 ng template DNA, 3.2 μL of 2.5 μmol/L primers, 2 μL 10× Buffer II, 0.1 μL polymerase, and distilled water to 20 μL.94°C 30 seconds
  30 cycles
    94°C 30 seconds
    60°C 20 seconds
    68°C 13 minutes
  Hold at 4°C
PrimeSTAR35 ng template DNA, 3.2 μL of 2.5 μmol/L primers, 4 μL 5× buffer, 1.6 μL dNTP, 0.4 μL Polymerase, and distilled water to 20 μL.30 cycles
    98°C 10 seconds
    68°C 10 minutes
  Hold at 4°C
LA Taq35 ng template DNA, 4 μL of 2.5 μmol/L primers, 2 μL 10× Buffer II, 3.2 μL dNTP, 0.2 μL polymerase, and distilled water to 20 μL.94°C 1 minute
  30 cycles
    98°C 10 seconds
   68°C 12 minutes
  72°C 10 minutes
  Hold at 4°C
KAPA 5–18 kb35 ng template DNA, 8 μL of 2.5 μmol/L primers, 2 μL 5× Buffer, 0.6 μL dNTP, 1.4 μL 25 mM Mgcl2, 0.2 μL polymerase(2.5 U/ μL), and distilled water to 20 μL94°C 30 seconds
  35 cycles
    94°C 20 seconds
    60°C 15 seconds
   68°C 12 minutes
  72°C 10 minutes
  Hold at 4°C
  QIAGEN
(0.1–10 kb)35 ng template DNA, 6.4 μL of 2.5 μmol/L primers, 2 μL Buffer, 1 μL dNTP, 0.2 μL enzyme, and distilled water to 20 μL.94°C 3 minutes
  35 cycles
    93°C 15 seconds
    62°C 30 seconds
    68°C 10 minutes
  Hold at 4°C
(>10 kb)70 ng template DNA, 6.4 μL of 2.5 μmol/L primers, 2 μL Buffer, 1 μL dNTP, 0.2 μL enzyme, and distilled water to 20 μL.94°C 3 minutes
  10 cycles
    93°C 15 seconds
    62°C 30 seconds
    68°C 13 minutes
  28 cycles
    93°C 15 seconds
   62°C 30 seconds
   68°C 13 minutes plus 20 seconds
  Hold at 4°C