What are possible reasons for reduced DNA yields with REPLI-g Kits?
Low yields with REPLI-g Kits for whole genome amplification (WGA) can result from a number of factors:
- inhibitors in the template DNA, e.g., phenol and SDS, EDTA > 1 mM
- low-quality, fragmented input DNA, e.g., degraded DNA from old samples, FFPE samples
- improper reaction conditions, e.g., wrong volumes pipetted, insufficient reagent mixing, denaturation and neutralization steps omitted, or carried out incorrectly
- Thermocycler incorrectly programmed, e.g., incubation time set incorrectly, reaction temperature too high (heated lid not adjusted to 70°C)
Note! When using a thermocycler model that does not allow adjusting the temperature of the heated lid, REPLI-g incubation temperature has to be reduced to 25-28°C to ensure optimal reaction conditions and amplification efficiency!