Why are DNA concentrations low/high after first PCR?

  • Updated

DNA-only protocol

The expected concentration after the first PCR for V2 Chemistry ranges from 0.2 – 2.0 ng/μL.

Low concentrations may be caused by:

  • A low cell number input during encapsulation (< 3,500 cells/μL for V2 chemistry, < 2,800 cells/μL for V3 chemistry)
  • A clog during cell barcoding that prevented Barcoding Beads from entering the cartridge
  • Used expired reagents
  • PCR settings were incorrect  – Use the correct ramp rate, temperatures, and annealing/extension times
  • Used the incorrect Ampure XP reagent/sample ratio
  • The sample and Ampure XP reagent were not at room temperature before clean up
  • 80% EtOH was not freshly prepared
  • Forgot the UV-light treatment of emulsions after Barcoding – Tapestri Instruments with serial numbers MBT-2019 or lower only
  • Used the wrong UV light during UV-light treatment of emulsions after Barcoding – Tapestri Instruments with serial numbers MBT-2019 or lower only

High concentrations may be caused by:

  • A high cell number input during encapsulation (> 4,000 cells/μL for V2 chemistry, > 3,200 cells/μL for V3 chemistry)
  • Excessive drop merging after Barcoding (amplification takes place in ‘quasi-bulk environment)
  • Excessive Barcoding Bead loading after Barcoding
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