3 Matching Annotations
  1. Feb 2023
    1. DNA fragmentation by restriction digestion prior to dropletgeneration enables optimal accuracy by separating tandemgene copies, reducing sample viscosity, and improving templateaccessibility for input samples >66 ng per well

      NEB says

      Digestion is recommended whenever DNA input is greater than 75 ng Source

      NEB says that biorad recommends these enzymes: AluI, CviQI, HaeIII, HindIII-HF, MseI

      More guidelines

      • Assemble ddPCR reactions at room temperature, this allows the restriction enzyme to digest the gDNA during the reaction set-up period
      • Prepare reaction mixes as you would for a standard ddPCR reaction. Add 0.5–1 μL of each restriction enzyme (5–20 units, depending on enzyme concentration) to the reaction mixture
      • After set-up, simply continue droplet generation as normal
      • Restriction enzyme will be inactivated during first PCR denaturation step
  2. Nov 2022
    1. For those shorter than 25 bases, a standard (dual labeled) BHQ™ Probe may be perfectly satisfactory.

      Nova might be better to have for 1-step ddPCR applications where the BHQ quenchers are unstable due to reduction of the N=N azo bond by the DTT additive