A High throughput DNA extraction method for plant tissues rich in polyphenols, terpenes and polysacc

Procedure

1. Samples of 2 to 5 mature Eucalyptus leaves or at least 10 pine needles were collected directly into seed pocket envelopes and kept on 3 - 4 changes of self-indicating silica gel in a sealed storage container until dried. Pine needles often benefit from a subsequent round of freeze drying to completely remove all moisture before grinding. A 6 mm single-hole punch was used to cut 5 discs from dried leaves of Eucalyptus and other species, equivalent to ~20 mg in weight.
2. Dried Acacia and poplar leaf was weighed out to ~20 mg. Pinus species having very large genomes compared to most plant species, and having very thick cuticular coatings, we therefore used 30 mg of dried needles to compensate for the low genome copy numbers.
3. Samples were loaded into the wells of 1.2 ml, 96-well storage blocks together with a 3 mm chrome steel ball bearing.
4. Blocks were sealed with appropriate 8-cap strips. The remaining leaf material was stored out of direct sunlight in envelopes, in airtight containers containing self indicating silica gel.
5. Blocks were loaded onto a mixer-mill (Retsch/Qiagen Tissue-lyser) and oscillated for 2 by 3 min pulses at 30 rps. Orientation of the blocks was reversed after the first 3 minute pulse to ensure even, very fine powdering of all samples.
6. 400 ul of lysis buffer warmed to 60ºC (200 mM Tris-HCL pH 6.5, 70 mM EDTA pH 8, 2 M NaCl, 2% PVP 40, 20 mM sodium metabisulphite, 1% detergent such as sodium N-lauroyl sarcosine or Triton-X, 24 mM MgCl₂/MgSO₄, 0.1% spermine and 0.1% spermidine) was added to each sample and caps replaced.
7. Blocks were vortexed to wet and mix the contents completely then placed in a 60ºC oven for 60 minutes. Blocks were transferred to ice and 400 ul of 10% PVPP (hydrated overnight with stirring in 10 mM TRIS pH 6.5, then autoclaved) was added to each well. Blocks were sealed with caps and mixed well by inversion then left to settle out on ice for 60 minutes before being spun down at ~4000 rpm for 30 minutes in a swing-out rotor at R/T. (For robotics: I recommend that PVP360 be tried as this will not block the tips of a robot. 10% PVP360 is prepared in just the same way as the PVPP slurry and forms a viscous fluid which should be easier for a robot to pipette once it has been calibrated to handle a viscous fluid. This would need to be determined for each robot type).
8. Using a hand-held 8 channel pipette or robotics, 400 ul of the supernatants was transferred to larger volume, 2.2 ml 96-well storage blocks, avoiding the compacted solid pellet as much as possible.
9. 5 ul of 10 mg/ml RNase A was added and the samples left at room temperature for 10 minutes.
10. The supernatants were precipitated with 200 ul of 10M ammonium acetate and 600 ul of isopropanol per well, blocks were sealed with a solvent resistant seal, inverted to mix then incubated at -20ºC overnight.
11. Precipitated supernatants were centrifuged at ~4000 rpm for 30 minutes at 10ºC and EtOH carefully drained away or aspirated.
12. DNA pellets were washed with 1 ml of ice cold 70% EtOH and again centrifuged as above for 10 minutes, drained or aspirated and air dried for 20 minutes.
13. Pellets were resuspended in 200 ul of 10 mM Tris Ph 8 and DNA again precipitated with 200 ul of 30% PEG8000/1.2 M NaCl on ice for 60 minutes, then spun down as before at ~4000 rpm for 30 mins at 10ºC.
14. PEG supernatants were carefully aspirated and DNA pellets were washed once with 1.5 ml ice-cold 70% EtOH then centrifuged at ~4000 rpm for 15 minutes and the wash carefully removed by draining or aspiration.
15. Blocks were air dried and DNA pellets resuspended in 200 ul of 10 mM Tris pH 8.
16. DNA was diluted into a PCR plate at 1 in 50 using 10 Mm Tris, pH 8 and kept at -20ºC as a working stock for PCRs, the remaining concentrated stock was stored at -20ºC, or lyophilised and stored at -80ºC indefinitely.