Genomic DNA from C. difficile and enterococci bacterial isolates were extracted from frozen pellets. Prior to extraction, the cell pellets were mechanically lysed in a PowerLyzer Homogenizer (Qiagen) with 0.1 mm glass beads. DNA was extracted using the DNeasy Blood and Tissue kit with QIAcube automation according to the manufacturer’s instructions (Qiagen). The 16S rRNA gene was amplified using universal 27F and 1492R primers. All PCR products were purified with the Monarch PCR & DNA Cleanup kit (New England Biolabs) and sequenced by Sanger DNA sequencing performed by CHOP’s Nucleic Acid PCR Core Facility. The first 50 bases and everything after 850 bases were trimmed from the 5’ end of each sequence and sequence analysis was carried out by NCBI BLASTn similarity search within the 16S Ribosomal database with an E-value cutoff of 0.01. Enterococci were identified on the parameters of highest percent identity and agreement between forward and reverse sequences.
Powerlyzer homogenizer
Manufactured by Qiagen
Sourced in United States
The PowerLyzer Homogenizer is a versatile laboratory equipment designed for efficient sample preparation. It utilizes high-speed mechanical disruption to homogenize a wide range of sample types, including tissues, cells, and other solid materials. The PowerLyzer Homogenizer is capable of processing multiple samples simultaneously, allowing for increased throughput and productivity in the laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Monarch pcr dna cleanup kit, by New England Biolabs (1 mentions)
Qiacube automation, by Qiagen (1 mentions)
Dneasy blood and tissue kit, by Qiagen (1 mentions)
Powersoil kit, by Qiagen (1 mentions)
Powersoil dna isolation kit, by Qiagen (1 mentions)
Multiskan go, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Dna free kit, by Thermo Fisher Scientific (1 mentions)
Anti mouse igg hrp, by Merck Group (1 mentions)
Urea, by Avantor (1 mentions)
Mouse anti pgk1 antibody, by Thermo Fisher Scientific (1 mentions)
Anti flag m2 hrp, by Merck Group (1 mentions)
Penta his hrp conjugate kit, by Qiagen (1 mentions)
6 protocols using powerlyzer homogenizer
1
Bacterial DNA Extraction and 16S Sequencing
2
Quantifying Bacterial Disease Progression
3
Extracting Genomic DNA from Diverse Marine Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Genomic DNA was extracted from 0.22 µM membranes of filtered seawater, sea grassss and gut fecal pellets from each of the four sea urchin species from reef water and seagrass biotopes (∼200 mg). We used the QIAGEN PowerSoil™ kit (QIAGEN LLC, Germantown Road, Maryland, USA), following manufacturer’s instructions with the following modifications: (1) gut content sample homogenization (3000 r.p.m. for 2 min at room temperature) in a PowerLyzer homogenizer (QIAGEN LLC, Germantown Road, Maryland, USA), and (2) Elution was performed using 100 µl of sterile PCR water previously warmed at 65 °C, to increase DNA yield, allowed to remain on the filter for 5 min incubation at room temperature before the final centrifugation step. A Qubit® dsDNA HS (High Sensitivity) Assay Kit was used to assess DNA concentration (ranging from 5–100 ng/ul) of purified extracts using the Qubit® Fluorometer at room temperature (Waltham, Massachusetts, USA).
4
Soil DNA Extraction Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For DNA extraction purposes, homogenized hizosphere soil samples (0.5–1 g) were pre-treated by vortexing for 1 h in 2 mL of sodium phosphate buffer (0.1 M, pH 8) and centrifuged at 16,000 × g for 10 min (He et al., 2005 (link)). The pellet was subjected to cell disruption by bead-beating for 30 s using a Powerlyzer® homogenizer (Mo-Bio Laboratories, Carlsbad, CA, United States), and the solution was subjected to DNA purification using Power Soil® DNA Isolation Kits (Mo-Bio Laboratories), according to manufacturer instructions. The quality and quantity of DNA extracts were measured using a microplate spectrophotometer (Multiskan GO, Thermo Fisher Scientific, Inc., Waltham, MA, United States). The DNA purity was assessed by determination of the A280/A260 absorbance ratios and only DNA extracts with absorbance ratios of ≥1.8 were used for bacterial community analyses.
5
RNA Extraction from Murine Spleen and Liver
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For RNA extraction, half of the spleens of 8–12‐week‐old female mice were homogenized in 1 mL of Trizol reagent (Invitrogen, Carlsbad, CA) using the PowerLyzer homogenizer (MO BIO Laboratories, Carlsbad, CA) with ceramic beads. For RNA extraction of the livers, approximately 0.1 g piece of the left liver lobe of 8–12‐week‐old male mice were homogenized. The liver RNA samples were further purified using the DNA‐free kit (Ambion).
6
Preparation of Total Cell Lysates
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For the preparation of total cell lysates in Fig. 6A , about 10 OD600 units of cells at midlog phase (OD600 = 0.8 to 1.0) were harvested in 1.5 mL tubes, flash frozen in liquid nitrogen, and stored at −80°C. The cells were treated using a cell wall loosening procedure (2 M LiAc on ice for 3 min followed by 0.4 M NaOH on ice for 3 min),21 (link) and disrupted in 50 mM Tris (pH 8.0), 1% SDS, 8 M urea (VWR, 28877), 2 mM DTT, 5 mM EDTA with glass beads at 3,000 rpm for 30 sec with PowerLyzer homogenizer (MO BIO Laboratories, 13155, Carlsbad, California, USA) at 4°C. One volume of 4X sample buffer was added to 3 volumes of the lysates and loaded onto the gel. Proteins were detected using a mouse anti-Pgk1 antibody (Invitrogen, 459250) with anti-mouse-IgG HRP (Sigma, A9917), or an anti-Flag M2-HRP (Sigma, A8592). For detecting the His-tagged human NRBF2 MIT domain, Penta-His HRP conjugate Kit (Qiagen, 34660) was used with BenchMark (Invitrogen, 10747012) as a marker.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!