Amplification of the V4 region of 16S ribosomal DNA (16S rDNA) (IDT, Coralville, IA) was performed in 50-μL reactions and then purified with both Agencourt XP DNA purification beads (Beckman Coulter, Beverly, MA) and QiaAmp DNA Mini Spin columns (Qiagen). The sequencing was performed using two-pair end MiSeq, and data were analyzed using QIIME software.21 (link) The trimmed reads were clustered into operational taxonomic units (OTUs) at 97% identity level over an alignment and assigned to the respective genus-level taxa. α Rarefaction was performed using Observed Species and Shannon index. β Diversity was estimated by computing unweighted UniFrac distances between samples using QIIME.22 (link) The relative abundance of each taxon was then computed for each mock community sample at each taxonomic level.
Qiaamp dna mini spin columns
Manufactured by Qiagen
The QIAamp DNA mini spin columns are designed for rapid and efficient purification of genomic DNA from a variety of sample types. The columns contain a silica-based membrane that binds DNA, allowing for the removal of impurities during the purification process. The purified DNA can then be used in various downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
3 protocols using qiaamp dna mini spin columns
1
16S rDNA V4 Amplification and Sequencing
2
Quantification of Cellular Nucleic Acids
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Levels of nucleic acids were analyzed by real time polymerase chain reaction (PCR). RNA and DNA were purified from cells and/or from subcellular fractions (ie cytosol and conditioned media) using RNeasy and QIAamp DNA mini spin columns (Qiagen) respectively. RNA was reverse transcribed into cDNA using the iScript Advanced cDNA kit (BioRad). DNA was amplified by qPCR using the iTaq Universal SYBR Green Supermix (BioRad) in an ABI Prism 7500HT sequence detection system (Applied Biosystems) with the relevant PCR primers (sequences provided in Schuliga et al (32) ). For RNA quantitation, the Downloaded from journals.physiology.org/journal/ajplung at University of Groningen (129.125.166.083) on September 26, 2021.
threshold cycle (CT) value determined for each gene of each sample was normalized against that obtained for 18S rRNA, used as an internal control. The level of mRNA for a particular gene is proportional to 2 -(ΔCT) , where ΔCT is the difference between the CT values of the target gene and 18S rRNA. Relative levels (2 -CT ) of mtDNA and nuclear DNA (nDNA) in the cytosol fraction and media were measured using PCR primers for the mitochondrial and nuclear genes, tRNA Leu(UUR) and β2-microglobulin (B2M) respectively, expressed as a percentage of their levels detected in denatured whole cell extracts.
threshold cycle (CT) value determined for each gene of each sample was normalized against that obtained for 18S rRNA, used as an internal control. The level of mRNA for a particular gene is proportional to 2 -(ΔCT) , where ΔCT is the difference between the CT values of the target gene and 18S rRNA. Relative levels (2 -CT ) of mtDNA and nuclear DNA (nDNA) in the cytosol fraction and media were measured using PCR primers for the mitochondrial and nuclear genes, tRNA Leu(UUR) and β2-microglobulin (B2M) respectively, expressed as a percentage of their levels detected in denatured whole cell extracts.
3
Isolating Mitochondrial DNA from Fibroblasts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Primary cultures of human lung fibroblasts established as previously described and grown to confluence in T75 tissue culture flasks were harvested by trypsinization (32) . Mitochondria were isolated using the Mitochondrial Isolation Kit for Cultured Cells (ThermoScientific) according to the Manufacturer's instruction. DNA was extracted from mitochondria using QIAamp DNA mini spin columns (Qiagen). mtDNA was isolated from fibroblasts rather than epithelial cells because high numbers of cells were required to isolate enough mtDNA for experimentation.
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!