Which DNA isolation protocols do you recommend for Illumina sequencing?

We recommend DNA isolations with spin-column kits/protocols that should include an RNase digestion step. Spin column kits have the advantage that they will reliably generate clean DNA samples that are fully dissolved and have fragment sizes longer than 10 kb (following protocol instructions precisely). Other protocols can work too, but the spin column protocols are the most commonly used ones and very reliable.
  • Spin column DNA isolation kits are available from multiple vendors including Qiagen, Zymo, Omega Biotek, Sigma, and Norgen Biotek e.g. Qiagen DNeasy Blood & Tissue kit with added RNAse A (RNase A 100 mg/ml; cat. no. 19101).
  • Some vendors also offer these kits in a 96-well spin-plate format for large sample numbers (e.g. Qiagen, Zymo).
  • Only use a protocol that includes an RNase digestion step to remove any contaminating RNA; RNA can inhibit the DNA sequencing library preparation.
  • Plant samples will require a dedicated kit that includes a lysis buffer designed to capture harmful plant chemicals like phenols (e.g. Qiagen DNeasy Plant). Without protective additives in the lysis buffers, plant chemicals will damage the DNA.
  • Similarly, soil samples are rich in inhibitors of enzymatic reactions. Dedicated protocols and kits that can remove such chemicals (e.g. DNeasy Powersoil Pro) and are highly recommended.
  • If accurate quantification of the resulting DNA samples is required, absolutely avoid any protocols that employ the chemical CTAB. Spin column protocols are usually CTAB-free.
  • To achieve the cleanest DNA isolation, only use at most half the sample amount of the maximum recommended by the manufacturer.
  • Spin-column isolation tips: perform the “optional” steps described in the manufacturers manual. Always perform at least two spin column washes (with the kit wash buffer) after binding of the sample to the column matrix. Add a short “dry spin” of the column after the washes and before the elution buffer addition to avoid any carryover of the ethanol wash buffer.  Extend the incubation times for elution of DNA samples from spin columns to at least 5 minutes – or perform two consecutive elutions instead.
  • NEVER use heparin as an anticoagulant for blood samples destined for DNA or RNA sequencing. EDTA (preferred) or citrate anticoagulants should be used. Heparin co-purifies with nucleic acids and inhibits multiple types of enzymes like polymerases and ligases.

DNA Sample QC:

  • After extraction the DNA sample purity has to be determined via spectrometry (e.g. Nanodrop). Please see the sample requirements page for the recommended values for your protocol. It is certainly helpful to also record the entire UV absorption spectrum as it provides additional information. For DNA samples the 260/230 nm ratio should be >2 and the 260/280 nm ratio 1.8-2.0.
  • To assess the DNA sample integrity and verify the removal of RNA, DNA samples should best be analyzed by agarose gel electrophoresis, see: How should I QC my genomic DNA samples before sequencing? Please email us an agarose gel electrophoresis image with the DNA samples. For spin-column protocols the DNA fragments should be longer than 10 kb or 15 kb. Shorter fragments indicate DNA damage before the DNA isolation; please inquire with us in such cases.
Please also see:

How should I QC my genomic DNA samples before sequencing?

How should I purify my samples? How should I remove DNA or RNA contamination?

Do you offer DNA isolations and RNA isolations as a service?

How do I prepare DNA samples for RR-Seq (reduced representation sequencing)?


Category: 03 Sample Preparation & Sample Requirements

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