A total of 19 culled deer captured in the summer of 2012, 2013, and the spring of 2016 were examined (Table 1 ). Previous work examined the plant species compositions in stomach contents of Yaku sika, and it was shown that, the occurrence of major plant species in deer diets were not significantly different between seasons (i.e. summer and winter)29 . Total rumen contents were collected into a 50-ml sterile tubes using sterile spoons and immediately placed on ice until transported back to the lab (within 5 days) where they were stored at − 80 °C. The rumen contents were freeze-dried (VD250-R; TAITEC Co., Saitama, Japan), homogenized in a coffee grinder (Russell Hobbs, Failsworth, UK) which was thoroughly washed and wiped with 70% ethanol after processing each sample, and stored at − 80 °C for molecular analysis.
Vd 250r
Manufactured by TAITEC
Sourced in Japan
The VD-250R is a laboratory vacuum drying oven designed for drying and dehydration processes. It features a digital temperature control system and a stainless steel interior chamber.
Automatically generated - may contain errors
5 protocols using vd 250r
1
Yaku Sika Deer Rumen Analysis
2
Characterization of Fresh and Black Garlic
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Fresh and black garlic were collected from Japan and abroad. In addition, three garlic accessions managed by Yamaguchi University were used in this study (Table 1 ). These accessions were obtained from local markets or national institutions in each country. Detailed information regarding these accessions was reported by Etoh [27 (link)] and Hirata et al. [28 (link)]. ‘White-roppen’ black garlic was processed in a rice cooker using warm mode for two weeks. The freeze-dried powder of fresh and black garlic bulbs was used as the experimental material. To prevent the loss of components, they were cut into thin slices on ice, immersed in liquid nitrogen for a few seconds, and then dried in a freeze-dryer (TAITEC vacuum freeze-drying equipment VD-250R) for 3 weeks. For each sample, 1 to 5 bulbs were prepared (Table 1 ).
3
PLGA Microparticle Synthesis by Emulsion Method
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PLGA (Mw 10-15 kDa 50:50, Akina AP041) was used to synthesize microparticles using oil in water single emulsion method. Briefly, 100 mg of PLGA was dissolved in DCM for 10 min. For some experiments, Cy5 (25 μg mL -1 ) or rif (50 mg) were added to PLGA-DCM solution. This organic phase was added to 10 mL of 1% PVA and homogenized at 10,000 rpm (for 2 μm particles) and 12,000 rpm (for 1 μm particles) for 2 min. For 500 nm particles, probe sonication (Q125, Q-sonica) was used at 30% amplitude with 10 s pulses for 2 min. The homogenized mixture was added to 100 mL of 1% PVA with constant stirring at 300 rpm for organic solvent evaporation. After 4-5 h, the particles were centrifuged at 10,000 g for 5 min and washed thrice with deionized water to remove residual PVA. The samples were snap-frozen and lyophilized (Taitec VD-250R) at -45 • C under vacuum. As PLGA will degrade in the presence of water, lyophilization is essential to remove residual water and allow long term storage of particles as powders. The particles were stored at -20 • C.
4
Pollen DNA Extraction and Purification
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First, pollen samples (0.5 g) were lyophilized using a lyophilizer freeze dryer VD-250R (TAITEC, Koshigaya, Saitama, Japan). After being ground at 1500 rpm for 2 min using a ShakeMaster NEO homogeniser (bms, Shinjyuku, Tokyo, Japan), DNA was extracted using the protocol of MPure Bacterial DNA Extraction Kit (MP Biomedicals, Irvine, CA, USA). DNA purification of the samples was performed using the MPure-12 Automated Nucleic Acid Purification System (MP Biomedicals, Irvine, CA, USA). Quality control of DNA extracts was conducted using Synergy H1 (BioTek, Winooski, VT, USA) and QuantiFluor dsDNA System (Promega, Madison, WI, USA).
5
Extraction and Purification of 32K Supernatant
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The 32K culture supernatant (50 mL) was dispensed in an eggplant flask and frozen at −30 °C. The frozen 32K culture supernatant was lyophilized overnight with a freeze-drier (VD-250R; Taitec Co., Ltd., Japan) and then dissolved in 5 mL of sterilized water. The concentrated 32K culture supernatant (5 mL) was desalted by dialysis (4 °C, 24 h) with a Spectra/Por 6 instrument (Spectrum Laboratories; diameter: 11.5 mm; membrane material: standard regenerated cellulose membrane [standard RC membrane], molecular weight: 3500 Da cutoff). Using 1 L of Milli-Q water, the external solution was changed three times. The desalted 32K culture supernatant (approximately 6 mL) was lyophilized overnight and then dissolved in 6 mL of sterilized water. To the suspension, 14 mL of 99.5% ethanol was added, and the mixture was allowed to stand overnight at −30 °C. The sample was centrifuged (4 °C, 13,000× g, 1 h); the supernatant (nonpolar fraction) was separated from the precipitate (polar fraction), and the former was discarded. The polar fraction was dissolved in 10 mL of sterilized water, and the ethanol was removed with a rotary evaporator. The whole polar fraction (approximately 10 mL) was frozen at −30 °C, lyophilized overnight, and dissolved in 50 mL of sterilized water.
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