Pelleted bacterial cells were resuspended in 200 μl of lysozyme/mutanolysin (LM) solution (1xTE buffer (pH 8.0) containing 15 mg/ml lysozyme (Sigma, Germany) and 500 U/ml mutanolysin (Sigma, Germany)) ([30 (link)]). The cell suspension was vigorously shaken for 45 min at 13.000 rpm using an Eppendorf shaker. Afterwards the suspension was transferred to a 15 ml falcon tube containing 50 mg of 0.1 mm zirconia beads (Roth, Germany) and 700 μl RLT lysis buffer (Qiagen, Germany). After vigorous vortexing for 3 min, the beads were removed by centrifugation (11.000 g, 2 min, RT). 470 μl of 100 % ethanol (Roth, Germany) were added and thoroughly mixed with the supernatant which was then applied in two steps to the Qiagen Spin Column. The subsequent RNA extraction procedure was performed according to the manufacturer’s instructions, including the on-column DNase I digestion with the Qiagen DNAse I kit.
0.1 mm zirconia beads
Manufactured by Carl Roth
Sourced in Germany
The 0.1 mm zirconia beads are a type of lab equipment used for various applications. They are made of zirconium oxide and have a diameter of 0.1 millimeters. The beads are designed for use in lab settings.
Automatically generated - may contain errors
Lab products found in correlation
Lysozyme, by Merck Group (1 mentions)
Spin column, by Qiagen (1 mentions)
Mutanolysin, by Merck Group (1 mentions)
Rlt lysis buffer, by Qiagen (1 mentions)
Dnase 1 kit, by Qiagen (1 mentions)
Lysis solution, by Norgen Biotek (1 mentions)
T4 ligase, by Thermo Fisher Scientific (1 mentions)
Rnase a, by Merck Group (1 mentions)
Pbad myc hisc, by Thermo Fisher Scientific (1 mentions)
Q5 high fidelity dna polymerase, by New England Biolabs (1 mentions)
Chloroform isoamylalcohol, by Carl Roth (1 mentions)
Roti phenol, by Carl Roth (1 mentions)
Sanger sequencing, by Eurofins (1 mentions)
Fastprep 24 instrument, by MP Biomedicals (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Nanodrop 1000 spectrometer, by Thermo Fisher Scientific (1 mentions)
Bioanalyzer, by Agilent Technologies (1 mentions)
Agilent rna 6000 nano kit, by Agilent Technologies (1 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (1 mentions)
Turbo dnase, by Thermo Fisher Scientific (1 mentions)
4 protocols using 0.1 mm zirconia beads
1
Microbial RNA Extraction Protocol
2
Bacterial RNA Extraction with Bead Beating
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The suspension of bacterial cells in the lysis solution (Norgen, Germany) was either transferred to the supplied bead tubes (unmodified protocol) or to bead tubes containing sterile 0.1 mm zirconia beads (Roth, Germany) (modified protocol); each tube contained 500 μl of ice-cold PCI-solution (see above). Bead beating was performed using the same parameters as described above. After centrifugation (1 min, 11.000 g, RT) the resulting upper aqueous phase was used for RNA extraction according to the Norgen protocol.
3
Cloning of E. coli asa Gene
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Overnight cultures of E. coli O157:H7 strain EDL933 were mechanically lysed by bead beating with 100 µl of 0.1 mm zirconia beads (Carl Roth, Germany) in a FastPrep-24 Instrument (MP Biomedicals). The genomic DNA was isolated with CTAB (Sigma Aldrich) and subsequent phenol-chloroform extraction using Roti®Phenol (Carl Roth, Germany) and Roti®Phenol plus chloroform/isoamylalcohol (Carl Roth, Germany). The RNA was subsequently removed using RNase A (20 mg/ml, Sigma Aldrich). The gene asa was amplified with PCR (primers: 8220+1F-NcoI, GAT CCA TGG GGA TGT TGC TGG TTT CAA ACA; 8220+245R-HindIII, GCC AAG CTT CTA TCT GTC TGC CGG AAT GG) by adding restriction enzyme cutting sites for NcoI and HindIII. For all PCRs, the Q5 High-Fidelity DNA Polymerase (New England Biolabs) was used. The vector pBAD-myc/His C (Invitrogen) and asa were double digested with NcoI and HindIII (Thermo Fisher Scientific) at 37 °C for 3.5 h and ligated using T4 ligase (Thermo Fisher Scientific). The ligation preparation was desalted by swimming filter dialysis and transformed in E. coli TOP-10 by electroporation. The successful cloning was verified by colony PCR (primers: pBAD-C+165F, CAG AAA AGT CCA CAT TGA TT; pBAD-C-R, TGA TTT AAT CTG TAT CAG GC) and Sanger sequencing (Eurofins, Ebersberg).
4
Comprehensive RNA and DNA Extraction
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In all protocols, RNA and DNA concentrations were either determined by the NanoDrop 1000 spectrometer (Thermo Fisher Scientific) and/or by the Qubit 2.0 Fluorometer (Invitrogen, life technologies) according to the Qubit RNA HS Assay Kits and Qubit dsDNA HS Assay Kit. Genomic DNA was isolated according to a CTAB (cetyltrimethylammonium bromide)-based protocol as described 44 (link). For RNA isolation, cell pellets were resuspended in 1 mL Trizol (Thermo Fisher) and mixed with approx. 0.2 mL 0.1-mm zirconia beads (Carl Roth, Germany). Cells were disrupted using the Ribolyzer (MP FastPrep) for bead beating (thrice at 6.5 m/s for 45 s). After incubation for 5 min at room temperature (RT), 0.2 volumes chloroform per mL Trizol were added and mixed for 15 s, followed by centrifugation at 12,000 × g at 4 °C for 15 min. The upper aqueous phase was transferred into new tubes and RNA was precipitated with isopropanol (Carl Roth). The RNA pellet was carefully dried at RT and solved in RNase-free water. Afterwards, DNA digestion was performed (Turbo DNase, life technologies). Complete DNA removal was tested with PCR using Taq DNA polymerase (Supplementary table S4 with primer rrsHR and rrsHF. Total RNA quality was checked using agarose gel electrophoresis and using the Bioanalyzer with the Agilent RNA 6000 Nano Kit (Agilent).
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