In this application note, researchers at Solis Bio-Dyne collaborated with researchers at MicroGEM to optimize a plant DNA extraction protocol to use MicroGEM's phytoGEM® extraction chemistry kit for plant applications with minimal sample size.

Using corn seed, corn leaf, tomato leaf, Cheniere rice seed, and apple leaf, the researchers used MicroGEM's phytoGEM temperature-driven reagent kit for plant DNA extraction. This is a novel use of the phytoGEM kit as it is typically used with MicroGEM's PDQeX Nucleic Acid Extractor. However, the PDQeX requires at least 10 μl of extraction volume. The researchers tested how moving the extraction reaction out of the PDQeX cartridge and into a 0.2 ml tube could provide increased extraction volume flexibility for applications using small sample sizes.

With a minimal input of 2 or 5 mg of leaf or seed sample, on average, over 5 ng/μl DNA was recovered from all samples.

The SolisFAST® SolisGreen® qPCR mix with UNG (no ROX) by Solis BioDyne was used to mitigate the potential presence of inhibitors in the starting materials and possible missed inactivation of the prepGEM enzyme after extraction. The combination of MicroGEM’s extraction and Solis-FAST® SolisGreen® qPCR amplification allowed the detection of all samples with 1 ng of input DNA at the 5.8S rRNA locus.

The researchers concluded the phytoGEM chemistry with low abundant samples, apart from the PDQeX platform, is able to provide large amounts of high-quality plant DNA suitable for downstream applications. Further, the combination of MicroGEM's temperature-driven extraction with SolisFAST SolisGreen qPCR Mix with UNG allows sample to qPCR results in less than 2 hours.

Overall, the optimized protocol allows:

  • fast, efficient plant DNA extraction
  • scalability depending on the sample size
  • flexibility depending on the number of samples
  • reduced plastic consumption and hazardous waste
  • less hands-on time
  • fast, sensitive, specific, and reproducible dye-based qPCR
  • consistent results with high-and low-copy targets
  • robust performance in the presence of PCR inhibitors
  • room temperature shipping and reaction set-up
  • minimized PCR carry-over contamination risk.

Read the app note here.