Library Quality Control
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      2. Library Preparation
      3. Library Quality Control

      PCR-Free Library Prep Quality Control

      Q: What quality control should I do after PCR-free library prep?

      A: Make sure to perform the following PCR-free library quality control steps:

      PCR-Free Libraries

      1. Determine the concentration (ng/µL) of each library using qPCR for accurate quantification.
      2. PCR-free libraries are not linear double stranded libraries, and due to the structure of the adapters on either end, the libraries will not run true to size and migrate differently on a system like the Bioanalzyer. We recommend to perform a PCR check to estimate the size of your PCR-free library, using primers that will bind and amplify S1/S2 sequences. We recommend the following PCR conditions using the QuantaBio sparQ Hi-Fi PCR master mix starting with 1 ng of PCR-free library:
      Reagent
      Stock Conc.
      Unit
      Final Conc
      Unit
      Volume to add for 1 rxn (uL)
      Nuclease-free water
       n/a
      n/a 
      n/a 
      n/a 
      19
      SparQ HiFi PCR Master Mix
      2
      X
      1
      X
      25
      Primer S1, 10 uM
      10
      uM
      0.5
      uM
      2.5
      Primer S2, 10 uM
      10
      uM
      0.5
      uM
      2.5
      PCR-free library
       variable
       ng/uL
       1
       ng/uL
      1
      Total
       
       
       
       
      50

      Program a thermocycler with conditions specified in the table below and the lid temperature set to 105 °C. Immediately pause the program when the block reaches 95 °C.

      STEP
      TEMP
      TIME
      CYCLES
      Initialization
      95°C
      2 min
      1
      Denaturation
      Annealing
      Extension
      95°C
      60°C
      72°C
      20 sec
      30 sec
      30 sec


      6
      Final Extension
      72°C
      5 min
      1
      Final Hold
      4°C
      Hold
      1

      Note that Singular Genomics (SG) non-indexed primers are 62 bp long, while SG indexed primers are 75 bp long. To calculate the expected library size, take the expected insert size and add 124 bp for non-indexed runs or 150 bp for indexed runs.

      Version: 6 Oct 2023