Thermomixer comfort

A 500 mg cell pellet is resuspended with 500 μl Cell Breaking Buffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 100 mM Tris-HCl pH8.0, 1 mM EDTA) and distributed in 280 μl aliquots. 200 μl Phenol:Chloroform:Isoamylalcool 25:25:1 and 300 μl 0.4–0.6 mm unwashed glass beads are added to each aliquot. Samples are vortexed for 10 min at 4°C using a Disruptor Genie from Scientific Industrial (US Patent 5,707,861). 200 μl TE are added to each lysate, which are then centrifuged for 5 min at 16100x g, 4°C. The upper layer (~400 μl) is transferred to a fresh tube, 2.5vol 100% EtOH are added and the sample mixed by inversion. DNA is pelleted for 5 min at 16100x g, 20°C. The supernatant is removed and the pellets resuspended in 200 μl RNAse A 250 μg/ml for 15 min at 55°C, 1000 rpm on a Thermomixer comfort (Eppendorf). 2.5 vol 100% EtOH and 0.1 vol NaOAc 3 M pH5.2 are added and the samples mixed by inversion. DNA is pelleted by centrifugation for 5 min at 16100x g, 20°C. The pellets are washed with 70% EtOH under the same conditions, the supernatant removed completely, and the pellets dried for 10 min at 37°C. The pellets are resuspended in a total volume of 100 μl water for 10 min at 55°C, 700 rpm on a Thermomixer comfort (Eppendorf).
DNA is run on a 0.6% 1X TBE agarose gel against a standard 1 kb GeneRuler, and quantified using Fiji. 500 mg cell pellet should yield 20–60 μg DNA.

Hydrolysis experiments with Dactylis glomerata grass were conducted in duplicates in a parallel assay. Fifty mM sodium acetate buffer, pH 5.0, was used in a total volume of 1 mL at 40 °C. The samples were shaken at 500 rpm in an Eppendorf Thermomixer comfort (Eppendorf AG, Hamburg, Germany). Prior to hydrolysis experiments, Dactylis glomerata grass was dried at 80 °C overnight. Substrate concentration was 5.0 mg/mL, and cellulase was added to a final protein loading of 2 µg/mg substrate. SWO1 was present at 0.02 µM, and the reference experiment used an equimolar amount of BSA instead of SWO1. Sampling was performed at suitable times. About 100 µL of the well-mixed supernatant was withdrawn and mixed with 100 μL of 100 mM NaOH to stop the reaction. Subsequently, the samples were centrifuged (13,000 rpm, 1 min, 25 °C), and the amount of released sugars in the cleared supernatant was assayed colorimetrically with the 3,5-dinitrosalicylic acid-based assay calibrated against glucose [77 (link)].

Individual ethanol-fixed cercariae were separated and placed in Eppendorf tubes (Axygen®, Sorenson BioScience, Salt Lake City, UT, USA), following which the ethanol was evaporated (Eppendorf ThermoMixer® Comfort; Eppendorf, Hamburg, Germany). DNA from each specimen was then isolated following lysis (using a guanidine thiocyanate lysis buffer) (Tkach and Pawlowski [24 ]). DNA was purified from the PCR mixture using Illustra™ GFX™PCR and Gel band purification kit (VWR International, Søborg, Denmark). DNA concentration and purity were measured using a Nanodrop 2000 spectrophotometer (Saveen & Werner ApS, Jyllinge, Denmark). The complete ITS1–5.8S–ITS2 regions of the rDNA were amplified using forward primer BD1 (5′-GTC GTA ACA AGG TTT CCG TA-3′) and reverse primer BD2 (5′-TAT GCT TAA ATT CAG CGG GT-3′) [25 (link)] under PCR cycling conditions as previously described [26 (link)]. Each sequence obtained from the PCR conducted was subjected to BLAST analysis at NCBI (National Center for Biotechnology Information). All sequences were submitted to GenBank and obtained accession numbers.