The 90:10 CMA:PEG400DA PCBAE gel films were cryomilled using the SPEX® SamplePrep 6770 Freezer/Mill® with 1% magnesium stearate. This PCBAE gel powder containing magnesium stearate was used to make the tablets used in this study. Particle size of the cryomilled gel powder was analyzed using a Shimadzu SALD-7101 nanoparticle size analyzer, with particles suspended in phosphate buffered saline (PBS). PBS was used as a blank to cancel the background signal. The powder suspension was stirred continuously using a built-in plate during the analysis.
Sampleprep 6770 freezer mill
Manufactured by SPEX
Sourced in United States
The SamplePrep 6770 Freezer/Mill is a laboratory equipment designed for cryogenic grinding and pulverization of solid samples. It utilizes a high-impact piston to grind samples while maintaining the sample at a low temperature, enabling efficient size reduction and homogenization of a variety of materials.
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Lab products found in correlation
Hand held saw, by Dremel (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Isopropanol, by Merck Group (1 mentions)
Dnase 1, by Qiagen (1 mentions)
Nanodrop spectrophotometer, by Agilent Technologies (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Nucleospin plant 2 kit, by Macherey-Nagel (1 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
4 protocols using sampleprep 6770 freezer mill
1
Cryomilled PCBAE Gel Powder Analysis
2
Ancient DNA Extraction and Sequencing
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DNA samples were collected from the 36 ancestors, with preference for petrous-temporal bones or molar teeth, and transported to the University of Tennessee-Knoxville (UTK) (Dataset S2 ) (9 ). Targeted regions of the bone were isolated using a Dremel handheld saw and the surfaces decontaminated prior to grinding using a SPEX SamplePrep 6770 Freezer/Mill. Between 0.2 to 0.3 grams of bone powder were used for DNA extraction as per Dabney et al. 2013 (57 (link)). Double-stranded DNA libraries were prepared using a modified NEBNext Ultra II library preparation protocol with partial USER treatment and quantified using a Bioanalyzer High Sensitivity DNA assay and quantitative qPCR (Applied Biosystems). Ancestors with high enough DNA concentration (n = 35) underwent whole genome enrichment (myBaits) and were test sequenced on an Illumina MiSeq at UTK’s Next-Gen Illumina Sequencing Core Facility. Of those, 31 were subjected to deeper sequencing using a NovaSeq (4 lanes) and HiSeq 4000 (2 lanes) at the University of Pennsylvania’s Next Generation Sequencing Genomics Core.
3
Fungal Total RNA Extraction and RNA-Seq
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Total RNA was extracted from 3 day and 7 day-old cultures as described [16 (link)]. In short, mycelia were harvested by filtering through Miracloth (Calbiochem), squeeze dried, snap frozen in liquid nitrogen and stored at -80°C until use. Frozen fungal pellets were ground using the SamplePrep 6770 FreezerMill (Spex). RNA was extracted from one hundred milligram of ground mycelium in 1 mL TRIZOL (Ambion). RNAs were precipitated with isopropanol (Sigma-Aldrich), treated with DNAse I (QIAGEN) and resuspended in 25 μL RNAse Free water. RNA purity and integrity were analyzed on NanoDrop Spectrophotometer and Agilent 2100 BioAnalyzer. For RNASeq, cDNA libraries were prepared using the TrueSeq RNA-Seq Sample Prep Kit V2 (Illumina Inc., San Diego, CA), and submitted to sequencing using Illumina 2x75 bp technology (Beckman Coulter Genomics). The RNA-seq data are available on NCBI's Gene Expression Omnibus and accession number GSE94878 [36 (link)].
4
Quantifying Fungal Biomass on Lignin PB1000
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Growth on the technical soda lignin PB1000 was followed by quantifying the fungal DNA material. Frozen mycelia were ground using a SamplePrep 6770 FreezerMill (SPEX® SamplePrep, Metuchen, NJ, USA). The biomass (20, 40, 60, 80 and 100 mg fresh weights) dry weights were determined after incubation for 24 h at 105 °C. This allowed the correlation between mycelial fresh and dry weights (Figure S1A ). Fungal DNA was extracted following the method previously described by Zhou [29 (link)]. NucleoSpin® Plant II kit (Macherey-Nagel, Dueren, Germany) was used according to the manufacturer’s instructions except for the changes described by Zhou [29 (link)]. A NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA) was used to determine the concentration of the extracted DNA. The conversion factors between the extracted fungal genomic DNA from negative controls not containing lignin and mycelium fresh weights were established (Figure S1B ). The calculated conversion factors were then applied to determine the dry weight from the extracted DNA for the samples containing lignin. Dry weight determination and DNA extractions were performed in triplicates for each selected weight.
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