Chelex

Macrozoobenthos invertebrates were collected and determined by M. Hess (Munich), nematodes were obtained as cultures from E. Schierenberg (Cologne), most fish material and some invertebrates were collected and/or determined by AN and RS, the Galaxias and Brachygalaxias samples by K. Busse (Bonn). F₁ Hybrids of Cottus were produced in the aquarium as described in Stemshorn et al. [42 ].
For most samples total DNA was extracted from ethanol preserved or fresh material using a standard Proteinase-K in SDS/EDTA buffer [tissue digestion in 500 μL HOM buffer (0.5% SDS, 100 mM Tris-HCl, 80 mM EDTA pH 8.0) and 5 μL Proteinase-K (20 mg/mL) for at least 3 h at 55°C; addition of 500 μL NaCl (4.5 M) and 300 μL Chloroform, gentle mixing for 15 min.; centrifugation for 10 min. at 10.000 rpm, transfer of upper phase (750 μL) without interphase in new tube; precipitation with 750 μL 99% Ethanol, gentle mixing and incubation at room temperature for 5 min., centrifugation for 10 min. at 13.000 rpm, removal of supernatant; 2× washing of the pellet with 500 μL 70% Ethanol, incubation at room temperature for 5 min., centrifugation for 10 min. at 13.000 rpm, complete removal of supernatant; airdried pellet dissolved in 100 μL TE buffer (10 mM Tris-HCl, 0.1 mM EDTA pH8.0)]. Alternatively, we used a standard CTAB buffer protocol [tissue digestion in 500 μL 2% CTAB buffer (2 g/100 mL CTAB, 1.4 M NaCl, 100 mM Tris-HCl, 20 mM EDTA, pH 8.0) and 15 μL Proteinase-K (20 mg/mL) for 1 h – overnight at 64°C; 2× extraction with Chloroform/Isoamylalcohol (24:1), gentle mixing for 10 min., centrifugation for 10 min. at 13.000 rpm, transfer of upper phase without interphase in new tube; precipitation with 400 μL 98% Ethanol, incubation at room temperature for 1 h, centrifugation for 20 min. at 13.000 rpm, removal of supernatant; 2× washing of the pellet with 500 μL 75% Ethanol, centrifugation for 10 min. at 13.000 rpm, complete removal of supernatant; airdried pellet solved in 50–100 μL TE]. For some samples we used also DNA isolated with a commercial kit (procedure according to the manufacturer; Qiagen, Düsseldorf) or released the DNA with a Chelex/Proteinase-K protocol (500 μL 5% Chelex suspension, 10 μl Proteinase K (20 mg/ml]) incubation 1 h to overnight at 64°C, 15 min. 95°C denaturation of Proteinase-K (important for the following RNAseA treatment). RNA was digested for all samples with RNAseA (10 mg/ml, Fermentas) before PCR reactions. We added to 50 μL DNA solution 2 μl Fermentas RNAseA and incubated for 1–3 h at room temperature.
Primers were designed according to partial or complete LSU rDNA sequences from GenBank for a variety of taxa, ranging from plathelminths, nematodes and arthropods to vertebrates. Primer sequences are listed in Figure 2.
PCR conditions were tested with temperature and MgCl₂ gradients. Amplification of LSU fragments were considerably enhanced by the addition of Q-solution (Qiagen, Düsseldorf) and single strand binding Protein (SSB, Sigma Aldrich). Final concentration of Q-solution is 1× (from 5× stock) and 1 μg SSB protein in a 20 μl PCR reaction mix.
The following PCR program was used to amplify the D1-D2 fragments: 4 min. at 94°C for initial denaturation; 45 cycles with 20 sec. 94°C, 20 sec. 52,5°C and 90 sec. 72°C, followed by 8 min. at 72°C for final extension. For most fragments we used the primer combination fw1 and rev1, some invertebrates amplified better with the combination fw1 and rev2.
In addition we amplified and sequenced a COI fragment currently applied in DNA barcoding applications for a sample of Cottus and the species of the Aphyosemion calliurum group (Cyprinodontiformes: Nothobranchiidae) for comparison with the LSU fragment. PCR primer for the amplification of the COI fragment for these taxa were taken from the literature (HCO-2198 [1 (link),43 (link)]) or designed according to published complete mitochondrial sequences and own data for the Cottus samples and the A. calliurum group. The following primers were used : Cottus COI forward: 5'-TTC TCG ACT AAT CAC AAA GAC ATT-3, Cottus COI reverse: 5'-TAG ACT TCA GGG TGA CCA AAG AAT CA-3, Aphyosemion forward: 5'-TAA GAA AAG GAT TTA AAC CT-3': "universal" reverse[43 (link)]: 5'-TAA ACT TCA GGG TGA CCA AAA AAT CA-3'.
All Aphyosemion and Cottus COI PCR reactions are done in 15 microliter reactions with the Qiagen Multiplex PCR Kit, including 3 microliter Q-Solution and 0.5 microliter of a 10 pmol solution of each primer. The following PCR program was used to amplify the COI fragments: 15 min. 95°C for initial denaturation and activation of the polymerase enzyme; 45 cycles with 20 sec. 94°C, 90 sec. 52°C and 90 sec. 72°C, followed by 8 min. at 72°C for final extension.
PCR products were checked on a 1.5 % agarose gel with ethidium bromide staining (130 V, 30–40 min.), cleaned with Millipore PCR cleaning plates and sequenced according to the manual with ABI BigDye Terminator ver.3.1 in both directions on an ABI 3700. Sequencing was done with the same primers as used in the PCR reaction for both gene fragments. Very long LSU sequences (> 1100 bp), especially if they contain GC rich stretches, were sequenced in addition with the internal primers fw2 and rev4. For sequencing it sometimes turned out to be helpful to increase the amount of template DNA to get better reads in difficult sequence regions.
Contigs were assembled with Lasergene SeqMan II (DNA-Star) and resulting sequences checked against GenBank for contamination. All contigs were checked by eye for ambiguous nucleotides in the regions sequenced for both strands. We counted positions with double peaks from one third up to same height in both strands to estimate the occurrence of different alleles or copies in the rDNA cluster.
The COI and LSU sequences for Cottus and Aphyosemion were aligned with Clustal X [44 (link)] and checked by eye with BioEdit 5.0.9 [45 ]. Aligned protein sequences were checked for a functional coding sequence to test against non-functional nuclear copies. A cluster analysis was done with the neighbour joining algorithm (NJ) as implemented in MEGA 3.1 [27 (link)]. We employed no model of sequence evolution and used p-distances to compare only the raw data without any assumptions on sequence evolution. Missing data or gaps were deleted in the pairwise comparison.
All sequences are deposited in Genbank under the accession numbers EF416965 – EF417284).

The pellet was resuspended in 100 μL of 6 % Chelex®100 (Bio-Rad Laboratories, Inc., USA). The suspension was incubated for 15 min at 56 °C and 1400 rpm, and subsequently heated at 99 °C for 8 min again at 1400 rpm. Both incubation steps were performed in a thermomixer. Finally, the samples were centrifuged at 16000×g for 2 min to precipitate food leftovers, cellular debris, and the resin.

An accurate mass (1.0 ± 0.002 g) of the Chelex-100 (anion exchanger) sorbent was homogeneously packed in glass column (10.0 cm length × 0.8 cm i.d). An aqueous solution of acetic acid (1.0 × 10⁻³ M) of pH 1-2 was introduced into the sorbent packed column and quartz wool was then placed at the top of the resin after the sorbent had established down. This step helps in avoiding the disturbance of the resin particles during percolation of the test solution. Column was then washed with water 2-3 times at a 2.0 mL/min flow rate. The test solution (25 mL) containing Y³⁺ and Sr²⁺ and DTPA (1.0 × 10⁻³ M) was permeated to pass through the column at a 2.0 mL min⁻¹ flow rate. Y³⁺ was only sorbed quantitatively, whereas Sr²⁺ species were passed through the column without sorption as specified from the radioactivity measurement of ⁹⁰Y³⁺ and ⁹⁰Sr²⁺ in the effluent. The sorbed Y³⁺ species were then recovered from the sorbent packed column with HNO₃ (10 mL, 1.0 × 10⁻¹M) at a 2.0 mL min⁻¹ flow rate. The recovered Y³⁺ solutions were heated to dryness, redissolved in ultra-pure water, and the Y³⁺ purity was finally determined via computation of the half life (t_1/2) as reported [39 (link), 44 ]. Moreover, the influence of other parameter such as flow rates (1.0–5 mL min⁻¹) and the internal diameter (0.8, 1.5, and 2.0 cm) on the analytical performance of Chelex-100 packed column for separation of ⁹⁰Y³⁺ from ⁹⁰Sr²⁺ was also examined.