Following the end points of 6 months for each cell line, harvested cell lines were faced to genomic DNA isolation using GeneJET Genomic DNA isolation kit (ThermoFisher, United States). Similarly, collected medium was centrifuged at 1,200 rpm and the collected pellet was used for genomic DNA isolation. For library preparation, barcode amplicons were amplified according to manufacturer’s guidelines provided by the Cellecta. Barcode sequences composed of 14 bp and 30 bp length variable nucleotides were sequenced on MiSeq Platform (Illumina, Inc.) using 150 bp pair-end method.
Genejet genomic dna isolation kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The GeneJET Genomic DNA Isolation Kit is a product that enables the extraction and purification of high-quality genomic DNA from a variety of sample types, including animal tissues, cultured cells, and bacterial cells. The kit utilizes a simple and efficient silica-based membrane technology to facilitate the rapid isolation of DNA.
Automatically generated - may contain errors
Lab products found in correlation
Miseq platform, by Illumina (2 mentions)
Sanger sequencing, by Thermo Fisher Scientific (1 mentions)
Dsdna br assay kit, by Thermo Fisher Scientific (1 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (1 mentions)
Neon electroporation device, by Thermo Fisher Scientific (1 mentions)
Genejet gel extraction kit, by Thermo Fisher Scientific (1 mentions)
Gel and pcr clean up system, by Promega (1 mentions)
8 protocols using genejet genomic dna isolation kit
1
Cell line genomic DNA isolation
2
Genotyping and Sexing Mouse Embryos
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Genomic DNA extracted from embryonic tail samples (GeneJet genomic DNA isolation kit, Thermofisher) was used to determine genotype via PCR using primers spanning the deltaE50-MD mutation site, followed by Sanger sequencing (GATC biotech). Sex of embryos was determined via PCR for the male-specific
SRY gene. Primer sequences are provided in
Table 1 . All trace data from sequencing results are available at the Figshare repository (see
Underlying data56 (link)).
SRY gene. Primer sequences are provided in
Underlying data56 (link)).
3
Constructing Microbial Community Standards
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All MCs were mixed in triplicate to account for pipetting error. These MCs ranged from 17–55 species in both equimolar and staggered compositions. One MC contained members at very low abundances of 0.1, 0.01 and 0.001% (
Dataset 1 ). Amplicons were generated either from cloned 16S rRNA gene amplicons, isolates available in the local culture collection of the Laboratory of Microbiology, Wageningen University, or strains ordered from
DSMZ and cultured according to DSMZ recommendations, after which genomic DNA was isolated using the Genejet genomic DNA isolation kit (Thermo fisher scientific AG, Reinach, Zwitserland). A 16S rRNA gene specific PCR was performed using the universal primers 27F (5’-GTTTGATCCTGGCTCAG) - 1492R (5’-GGTTACCTTGTTACGACTT) to obtain full length amplicons of which size and concentration were checked on a 1% agarose gel and which were column purified and quantified with the Qubit 2.0 fluorometer, and dsDNA BR assay kit (Invitrogen, Eugene, USA). Amplicons were mixed in the MCs to obtain the specified relative abundances. High quality full length reference sequences of all MC members were obtained by Sanger sequencing at GATC Biotech AG (Constance, Germany) with sequencing primers 27F - 1492R in order to confirm their identity. The MCs were diluted 10
3-fold and subsequently used as templates in PCRs for the generation of barcoded PCR products.
3-fold and subsequently used as templates in PCRs for the generation of barcoded PCR products.
4
CRISPR Murine Cell Line Editing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Murine TC lines B16F1-LacZ and LLC-LacZ were transfected with single guide RNAs (sgRNAs; Table S4 ) and recombinant Cas9 using the Neon electroporation device (Thermo Fisher Scientific) according to manufacturer’s instructions. For detailed settings, see Table S5 . Cells were subsequently plated in fresh culture medium. After 3 d, single-cell suspensions were seeded. Single-cell clones and the polyclonal pool were analyzed by PCR-based genotyping. In brief, genomic DNA was isolated using GeneJET Genomic DNA isolation kit (Thermo Fisher Scientific), and exon 5 was PCR amplified. PCR amplicons were excised, gel extracted using GeneJET Gel Extraction Kit (Thermo Fisher Scientific), and verified by Sanger sequencing.
5
Alzheimer's Disease Tissue Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Brains were dissected on ice and left hemispheres were flash frozen in liquid nitrogen for immunohistochemical analysis while the right was flash frozen in liquid nitrogen then stored at -80°C for cytokine array, immunoblotting, and Aβ ELISA. Based upon the importance of the temporal cortex as a region of AD-associated pathology, we elected to collect right temporal cortex regions for homogenizing in the cytokine array analysis buffer (RayBiotech, Norcross, GA). A portion of the supernatant was used for cytokine arrays. The remaining supernatant and the pellet were again homogenized but now in radioimmunoprecipitation assay buffer (RIPA, 20mM Tris, pH 7.4, 150mM NaCl, 1mM Na3VO4, 10mM NaF, 1mM EDTA, 1mM EGTA, 0.2mM phenylmethylsulfonyl fluoride, 1% Triton, 0.1% SDS, and 0.5% deoxycholate) with protease inhibitors (AEBSF 1mM, Aprotinin 0.8μM, Leupeptin 21μM, Bestatin 36μM, Pepstatin A 15μM, E-64 14μM). The supernatant was collected, protein concentration determined by Bradford protein assay [13 (link)], and stored at -80°C for immunoblotting and Aβ ELISA. Parietal cortices were isolated for DNA extraction using a Genejet Genomic DNA isolation kit (Thermo Fisher Scientific Grand Island NY), for 5-methylcytosine ELISA.
6
Bacterial 16S rRNA Gene Sequencing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The genomic DNA of all the bacterial strains was extracted using GeneJet Genomic DNA Isolation Kit (@Thermo Scientific Waltham, USA) according to the mentioned protocol. The 16S rRNA genes of the bacterial strains were amplified using universal primer pair 27F (5´ -AGAGTTTGATC-MTGGCTCAG- 3´) and 1492R (5´ -GGTTACCTTGTTAC-GACTT- 3´), respectively by PCR. The amplified PCR products were visualized under a UV transilluminator and were purified from the bands (approx;1500 bp) using Gel and PCR Clean-Up System (Promega, USA). The bacterial species were determined by 16S rRNA genes sequencing. The obtained forward and reverse sequences were joined together in the DNASTAR program. The final sequences were BLAST to retrieve the identical bacterial sequences. All the sequences were then aligned in CLUSTALW. The phylogenetic tree was made using MEGA X program (version 10.1.7) with 1000 bootstraps. The Neighbor-Joining method was followed to study the evolutionary relationships between our bacterial isolates sequences and all the retrieved sequences [23 (link)].
7
Barcode Sequencing of Dabrafenib Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Both end points after 3 months of dabrafenib treatment and harvesting of used medium samples were centrifuged at 1200 rpm to generate pellets. Pellets were then processed for genomic DNA (gDNA) isolation using GeneJET Genomic DNA isolation kit (ThermoFisher, USA). For amplicon NGS library preparation, Cellecta’s manufacturer guidelines were followed whereby amplification of barcode sequences was carried out via amplicon PCR using forward and reverse primer sequences. Finally, 14 bp and 30 bp length variable barcode sequences were sequenced on MiSeq Platform (Illumina, Inc.) using 150 bp pair-end method.
8
Detecting Dipylidium caninum in Dogs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Ninety-nine dogs were sampled in villages under field conditions in the area surrounding Bloemfontein (Free State, Republic of South Africa). The sampling procedure entailed the gentle swabbing of the anal region (including surrounding hair) with a sterile cotton swab. Swabs were collected from dogs living with their owners and stored at room temperature until processing for DNA isolation using the GeneJet Genomic DNA isolation kit (Thermo Fisher Scientific), according to the manufacturer’s recommendation for tissue samples. PCR based detection of D. caninum DNA obtained from 99 anal swabs from dogs resulted in successful amplification of the target region.
During the study, the anal area and hair surrounding it, as well as the sleeping area of the dog were checked for signs of proglottids. Proglottids were expelled by 12 animals at the time of swabbing. The swabs were subjected to PCR analyses, and if positive for D. caninum DNA (Table 3 ), the dogs were individually placed in kennels at Clinvet and screened for signs of proglottids.
During the study, the anal area and hair surrounding it, as well as the sleeping area of the dog were checked for signs of proglottids. Proglottids were expelled by 12 animals at the time of swabbing. The swabs were subjected to PCR analyses, and if positive for D. caninum DNA (
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!