The following procedure describes a column-based approach for the purification and precipitation of nucleic acid, which is recommended for Next Generation Sequencing applications, such as DNA-seq or RNA-seq.
DNA isolation (coupled with the Qiagen DNeasy Plant Mini Kit):
Collect the upper aqueous phase (600 µl), add 1.5 volume of buffer AW1 to the lysate, and mix it immediately by pipetting. Do not centrifuge the lysate at this stage. For example, to 600 μl supernatant, add 900 μl buffer AW1.
Transfer the mixture (~ 600 μl each time) into a DNeasy Mini spin column (white), including any precipitate. Centrifuge the samples at 8,000 g for 30 s, and discard the flow-through. Repeat the step with the remaining lysate.
Place the DNeasy Mini spin column into a new 2 ml microcentrifuge tube, add 500 μl buffer AW2 and centrifuge at 8,000 g for 30 s. This step may be repeated more than once, especially if the spin column membrane is not clean.
Transfer the DNeasy Mini spin column to a 1.5 ml microcentrifuge tube, and pipet 30 μl RNAse-free water directly onto the DNeasy membrane. After about 5 minutes, centrifuge at 5,000 g for 2 min to elute DNA.
To eliminate any remaining RNA, treat the samples with 2 μl RNase I at room temperature for 10 min.
RNA isolation (coupled with the Qiagen RNeasy Plant Mini Kit):
Collect the upper aqueous phase (600 µl), add 0.5 volume of ethanol (100%) to the lysate, and mix it immediately by pipetting. Do not centrifuge the lysate at this stage. For example, to 600 μl supernatant, add 300 μl ethanol.
Transfer the mixture (~ 600 μl each time) into an RNeasy spin column (pink), including any precipitate. Centrifuge the samples at 8,000 g for 30 s, and discard the flow-through. Repeat the step with the remaining lysate.
Place the RNeasy spin column into a new 2 ml microcentrifuge tube, add 700 μl buffer RW1 and centrifuge at 8,000 g for 30 s.
Add 500 μl buffer RPE to the RNeasy spin column and centrifuge at 8,000 g for 30 s to wash the spin column membrane (ensure that ethanol is added to the buffer). This step may be repeated more than once, especially if the spin column membrane is not clean.
Transfer the RNeasy spin column to a 1.5 ml microcentrifuge tube, and pipet 30 μl RNAse-free water directly onto the RNeasy membrane. After about 5 minutes, centrifuge at 5,000 g for 2 min to elute RNA.
To eliminate any remaining DNA in the samples, treat the samples with DNAase I according to the manufacturer's instructions.