Chloroform isoamyl alcohol 24 1

50 mL Falcon Tubes
RNAse A (Sigma Cat No. R6513)
Polyvinylpyrrolidone (PVP) (Sigma Cat No. PVP10) (not required)
Chloroform: Isoamyl alcohol 24:1 (Sigma Cat No. C0549)
Liquid nitrogen
β-mercaptoethanol (Sigma Cat No. 63689)
Trizma base (Sigma Cat No. 1503)
Ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA) (Chem Supply Cat No. EA023)
Agarose (Amresco Cat No. 0710)
Sodium Chloride (NaCl) (Ajax Finechem Cat No. 1103414)
Hexadecyltrimethylammonium bromide (CTAB) (Sigma Cat No. 52365)
EcoRI (not required- used for quality assurance) (NEB Cat No. R0101S)
HindIII-HF (not required- used for quality assurance) (NEB Cat No. R3104S)

Approximately 30 to 100 mg of glebal tissue from dried herbarium material or fresh samples were placed in 1.5 mL Eppendorf tubes. Two sterile metal beads were added to each tube. The tubes were subsequently submerged in liquid nitrogen for 5–10 s, and immediately after that homogenization of material was carried out using a Qiagen Tissue Lyser II (Retsch, Haan, Germany) for 3 min at 30 Hz. After homogenization, CTAB and SDS (10%) extraction buffers were added directly onto the crushed material, then Eppendorf tubes were incubated in a water bath for 12 h at 65 °C. In the extraction DNA enrichment procedure, phenol:chloroform:isoamyl alcohol 25:24:1 (Sigma-Aldrich Co., St. Louis, Missouri, USA) was used first, and chloroform-isoamyl alcohol 24:1 (SEVAG) solution thereafter. Isolated DNA was cleaned by washing with ≈100% isopropanol (Sigma-Aldrich Co.) and 70% ethanol (Sigma-Aldrich Co.), and then dried. The dried DNA was diluted in 50 µL 0.1 M TE buffer and 1 µL of RNase A (1 mg/mL) (Sigma-Aldrich Co.) was added. The concentration of obtained DNA was measured with a NanoDrop^® ND-1000 spectrophotometer (NanoDrop Technologies, Montchanin, DE, USA) and adjusted to an average value of 20 ng/μL for downstream applications.