FAQ - Genomic DNA Isolation

  • What are the yields of genomic DNA from different samples?
    • PrestoSpin D Bug - Bacteria 5 to 80 µg (1-5 ml culture), depending on the species
    • PrestoSpin D Fungi - Yeasts and fungi 5 to 7 µg/ml (yeast) or per 250 mg mycelium
    • PrestoSpin D Blood&Cell - Blood 2 to 4 µg/100 µl
    • PrestoSpin D Blood&Cell - Cell culture approx. 4 µg/106 cells
  • What are the maximum amounts of samples that can be processed?
    • PrestoSpin D Bug - Bacteria: 2 x 1010 cells corresp. to 5-6 ml E. coli
    • PrestoSpin D Fungi - Yeasts: 1-2 ml (O.D.600nm > 10)
    • PrestoSpin D Fungi - Fungi, mycelium: 250 mg wet weight
    • PrestoSpin D Blood&Cell - Human blood: 100-200 µl
    • PrestoSpin D Blood&Cell - Cell culture: 107 cells
  • Are highly viscous solutions a problem?
    No. With other kits using silica membrane mini spin and gravitational flow anion exchange columns, viscous solutions pose severe problems because of clogging of the matrix. PrestoSpin columns are highly porous and have a constant flow-through also at increased viscosity.
  • At which points can the DNA isolation procedure be interrupted?
    In principle at any point of the protocols. The reason is that Molzym developed buffers that prevent the degradation of nucleic acids even in crude extracts. Also, incubation times are optimised, and longer periods do not reduce the quality and quantity of isolated DNA.
  • Why is genomic DNA eluted with heated buffer EB?
    During the purification process, bound DNA is dehydrated by alcohol-containing washing buffers (WB, 70% ethanol). Rehydration before elution of large genomic DNA molecules is necessary and enhances at increased temperature.
  • Does elution buffer EB interfere with downstream applications?
    Buffer EB, used for elution of nucleic acids from the column, corresponds to buffer TE (1 mM EDTA, 10 mM Tris-HCl). Generally, this buffer does not interfere with downstream applications. However, in cases of a high proportion of template DNA to other components in PCR and sequencing reactions, a 1:4 dilution of buffer EB is recommended.
  • Can buffers other than EB used for elution of nucleic acids?
    Small amounts of complexing agents (EDTA) are needed for optimal yields. Diluted buffer EB (with sterile, deionised water) can be used with negligible loss of yield. Such diluted buffer eluates can be used directly for sequencing and transfection. If water is desired as an eluant, yields are somewhat reduced (by approx. 10-20%).

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