Application Note
Introduction
Materials and Methods
The workflows for both manual and automated extractions are shown in Figure 1. Resulting purified DNA was quantified via Promega’s QuantiFluor® dsDNA system and normalized per mg of input plant material. The quality of the purified DNA extracted from corn samples using silica spin column-based kits from Omega Bio-tek (D2411) and Company Q was assessed by real-time PCR. Briefly, undiluted, 10-fold and 100-fold dilutions of purified DNA were isolated and amplified using Agilent’s Brilliant III 2X SYBR® mix and corn-specific primers following a standard amplification protocol on the ABI 7900.
Results
The DNA yields obtained following manual protocols using Omega Bio-tek’s kit was significantly higher than the leading competitor’s kit on all 5 different plant samples tested (Figure 2). Table 1 shows the average Ct values obtained on serial dilutions of purified DNA from corn using both the kits. On average across the dilutions, the Cts with the Omega extraction were lower by ~4.9 cycles compared to Company Q’s. The Ct values suggest that there is at least 16x more DNA using the Omega kit over Company Q’s kit. Also, the ΔCt values with the Omega kit were closer to 3.3 that of Company Q (~2.94 vs. ~1.87 difference between 10-fold and undiluted) suggesting less qPCR inhibition in spite of more template DNA that seems to have been present. The results from the qPCR indicate that Omega Bio-tek’s kit was not only able to extract more DNA, but was also able to remove potential plant-related inhibitors that can hamper various downstream applications.
Twenty-three of the top agricultural and biofuel crops were subjected to automated extraction using Omega Bio-tek’s M1130 kit on Qiagen’s BioSprint® 96 and compared to the manual protocol of Company Q’s.
The results shown in Table 2 reveal that the automated methods resulted in significantly higher recoveries of DNA than the competitor’s manual method of 21 of the 23 plants tested. Because the extraction methods being tested are based on different chemistries, real-time PCR for the beta-tubulin gene was performed on a subset of 100-fold dilutions of plant extracts from both the methods. The differences in the resulting real-time PCR cycle threshold values were consistent with the amount of template measured to be present in each extract, or when adjusted to contain equivalent (1 ng) amounts of template (data not shown). These results indicate that matrix effects have not substantially influenced reported DNA recovery results.
Conclusions
Omega Bio-tek’s spin column and magnetic bead-based approaches for plant DNA extraction performed significantly better than its competitor. Overall, automated methods were much faster and required much less hands-on time than either of the manual methods. Up to 96 samples if 50 mg wet tissue (or 15 mg dry tissue) can be processed in parallel in less than 1 hour using Omega Bio-tek’s automated approach. Omega Bio-tek has leveraged their plant extraction experience and expertise to develop a robust solution for plant DNA extraction needs. Options are available for automated on open liquid handling and magnetic processor platforms, such as the Hamilton Microlab® STAR™, Hamilton Firefly® NIMBUS 96, and the Thermo KingFisher™ Flex®.
