Viral DNA was obtained from cellular extracts or supernatants using UltraClean BloodSpin DNA Isolation Kit (Mo Bio Laboratories, Carlsbad, CA) according to the manufacturer’s instructions. Adenoviral DNA content was quantified by qPCR using SYBER Green I Master plus mix (Roche Diagnostics, Basel, Switzerland) and the primers set 5 (Table S2 ). Viral DNA is expressed as relative to the cellular DNA content using the albumin 12 intron primers (primers set 6 in Table S2 ).
Ultraclean bloodspin dna isolation kit
Manufactured by Qiagen
Sourced in United States
The UltraClean BloodSpin DNA Isolation Kit is a laboratory equipment product designed for the rapid and efficient isolation of DNA from whole blood samples. The kit utilizes a proprietary spin-column technology to extract and purify DNA, ensuring high-quality genomic DNA suitable for downstream molecular biology applications.
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Lab products found in correlation
Syber green 1 master plus mix, by Roche (2 mentions)
Sybr green 1 master mix, by Roche (2 mentions)
Viia 7 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Ultrapure agarose, by Thermo Fisher Scientific (1 mentions)
Taqman snp assay, by Thermo Fisher Scientific (1 mentions)
Vacutainer, by BD (1 mentions)
Hiseq x ten platform, by Illumina (1 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (1 mentions)
Pcr master mix, by Qiagen (1 mentions)
Lightcycler 480 2, by Roche (1 mentions)
Sybr green 1 master plus mix, by Roche (1 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (1 mentions)
Hiseq x ten system, by Illumina (1 mentions)
Massarray platform, by Agena (1 mentions)
13 protocols using ultraclean bloodspin dna isolation kit
1
Viral DNA Isolation and Quantification
2
Genotyping of Diabetes-associated Variants
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Genomic DNA was obtained by taking 6 ml peripheral blood through arm phlebotomy in glass EDTA-tubes (Vacutainer®; BD Biosciences, Franklin Lakes, NJ, USA) from each patient. Patients were fasted at the time of blood sampling. Genomic DNA was isolated from 200 µl total blood, using the UltraClean® BloodSpin® DNA Isolation kit (Mo Bio Laboratories, Inc., Carlsbad, CA, USA), and the DNA-extraction protocol was performed according to the manufacturer's instructions. DNA quality was achieved by electrophoresis in Invitrogen 1% agarose gels (UltraPure™ Agarose; Thermo Fisher Scientific, Inc., Waltham, MA, USA), at 100 V over 50 min. DNA concentration was measured by spectrophotometry (Jenway 7305; Cole-Parmer Ltd., Staffordshire, UK). The KCNJ11 rs5219 (E23K), ABCC8 rs757110 (S1369A) and rs1799854 (−3C/T) polymorphisms were determined using 20 ng total genomic DNA per reaction, by allelic discrimination via quantitative polymerase chain reaction (qPCR) using a ViiA™ 7 Real-Time PCR system and TaqMan® SNP assays (Applied Biosystems; Thermo Fisher Scientific, Inc. Waltham, MA, USA) using the standard cycling conditions as follows: An initial denaturation stage 95°C for 10 min, 40 cycles of denaturation 95°C for 15 sec, and annealing at 60°C for 1 min, and post read stage or final extension at 60°C for 30 sec.
3
Adenoviral DNA Quantification Protocol
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Viral DNA was obtained from supernatants, cellular extracts or frozen tissues using the UltraClean BloodSpin DNA Isolation Kit (Mo Bio Laboratories, Carlsbad, CA, USA) according to the manufacturer's instructions. Viral genomes were determined by real-time qPCR using the SYBER Green I Master plus mix (Roche Diagnostics) and the primers Hexon Fw, 5′-GCCGCAGTGGTCTTACATGCACATC-3′ and Hexon Rv, 5′-CAGCACGCCGCGGATGTCAAAG-3′. Adenoviral copy number was relativized to the cellular DNA content using the albumin intron 12 primers of Fw, 5′-CTGTCATCTCTTGTGGGCTGT-3′ and Rv, 5′-GGCTATCCAAACTCATGGGAG-3′.
4
Fecal DNA Extraction and Quantification
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A Stool DNA Isolation Kit (Norgen, Biotek Corporation) was used for extracting DNA from a 300 μL aliquot of the previously homogenized feces samples following the manufacturer's instructions. The aliquot of the sample was obtained just after sowing for SCM, deposited in sealed containers and processed within the maximum time limit established for storage (72 h). The commercial kit used was selected for its high performance for the recovery of DNA from feces, despite the complexity represented by this sample source (Mathay et al., 2015 (link); Sanchez et al., 2017 (link)). Extracted DNA was recovered in 100 μL elution buffer included in the kit then stored at −20°C until being used. Regarding control strains, an Ultraclean BloodSpin DNA Isolation kit (MoBio Laboratories) was used for extracting DNA from the cell biomass recovered in 1X PBS, according to the manufacturer's instructions, eluting in the same final volume (100 μL). A NanoDrop2000 (Thermo Scientific NanoDrop Products) was used for spectrophotometric quantification of the DNA extracted from the control strains and then stored at −20°C for later assays.
5
Whole-Genome Sequencing of Isolates
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DNA was extracted using an Ultraclean Blood Spin DNA Isolation kit (MoBio Laboratories, Carlsbad, CA, USA) following the manufacturer’s instructions. The WGS of the selected isolates was carried out by Novogene Bioinformatics Technology Co., Ltd. (Beijing, China) using the Illumina HiSeq X-TEN platform. The quality control, assembly and genome identification can be retrieved in Supplementary Text S1 .
6
DNA Extraction from Dog Blood and Ticks
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The DNA extraction from dog blood samples was performed individually, while DNA extraction from ticks was performed by making 60 pools of 5 ticks each taken randomly from dogs with a positive result to any of the pathogens studied. To form the pools, female and male ticks, both adults, were included, as it was the most observed stage. Tick pools were homogenized to perform DNA extraction. In both cases, tick pools and dog blood samples, the extraction was performed using the UltraClean BloodSpin DNA Isolation Kit® (MoBio Laboratories Inc., Carlsbad, CA, USA; Cat. No. 12200-S) following the manufacturer’s protocol.
7
Genotyping of IFNλ3/4 SNP rs12979860
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Fasting venous blood (4 mL) was collected from the patients with SLE and controls using ethylenediaminetetraacetic acid (EDTA-2k)-treated tubes for DNA isolation.
The genomic DNA from patients and controls was extracted from peripheral blood samples using an Ultra Clean Blood Spin DNA Isolation Kit (MoBio Laboratories, United States) following the manufacturer’s instructions. The DNA concentration was determined using a NanoDrop 1000 (Thermo Fisher Scientific, Waltham, MA, United States). Samples with 260/280 ratios ≥1.8 were accepted for genotyping, and the genomic DNA was diluted to 12.5 ng/μL and stored at −20°C until use. The SNP rs12979860 (C/T) was detected by real-time polymerase chain reaction (qPCR) conducted on a LightCycler 480 II (Roche, Rotkreuz, Switzerland) in a total reaction volume of 5.0 μL. The qPCR was performed with 12.5 ng of DNA, 2.5 μL PCR Master Mix (Qiagen, Hilden, Germany), 0.1 μL TaqMan Probes (C_7820464_10 for rs12979860), and 0.4 μL DNase-free water. The allelic discrimination plots for the SNPs in the IFNλ3/4 locus were determined to genotype each sample using LightCycler®480 Genotyping Software.
The genomic DNA from patients and controls was extracted from peripheral blood samples using an Ultra Clean Blood Spin DNA Isolation Kit (MoBio Laboratories, United States) following the manufacturer’s instructions. The DNA concentration was determined using a NanoDrop 1000 (Thermo Fisher Scientific, Waltham, MA, United States). Samples with 260/280 ratios ≥1.8 were accepted for genotyping, and the genomic DNA was diluted to 12.5 ng/μL and stored at −20°C until use. The SNP rs12979860 (C/T) was detected by real-time polymerase chain reaction (qPCR) conducted on a LightCycler 480 II (Roche, Rotkreuz, Switzerland) in a total reaction volume of 5.0 μL. The qPCR was performed with 12.5 ng of DNA, 2.5 μL PCR Master Mix (Qiagen, Hilden, Germany), 0.1 μL TaqMan Probes (C_7820464_10 for rs12979860), and 0.4 μL DNase-free water. The allelic discrimination plots for the SNPs in the IFNλ3/4 locus were determined to genotype each sample using LightCycler®480 Genotyping Software.
8
Adenoviral Transduction of Tumor Spheres
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CP15 spheres were tripsinized as single cells, seeded at a density of 6 × 103 cell per well in poly-HEMA coated plates and infected with Adwt, or AdNuPAREmE1A at 1000 vp/cell. At 72 h post-infecction, 25% of the supernatant of infected cells was used to infect a new passage of tumorspheres. This was performed for 3 passages. Viral genome quantification was performed for all the passages. Viral DNA was obtained using the UltraClean BloodSpin DNA Isolation kit (Mo Bio Laboratories, Carlsbad, CA) according to the manufacturer´s instructions.
DNA from frozen liver tissue was obtained by incubating in a lysis buffer (100 mM NaCl, 10 mM TrisHCl pH8.0, 25 mM EDTA, 0,5% SDS) containing 0,1 mg/mL proteinase K overnight at 55°C, and then purified by phenol-clorophorm method.
Viral genomes were determined by qPCR using SYBR Green I Master mix (Roche Diagnostics, Basel, Switzerland) and the hexon primers (Supplementary Table 1 ) as described [39 (link)].
DNA from frozen liver tissue was obtained by incubating in a lysis buffer (100 mM NaCl, 10 mM TrisHCl pH8.0, 25 mM EDTA, 0,5% SDS) containing 0,1 mg/mL proteinase K overnight at 55°C, and then purified by phenol-clorophorm method.
Viral genomes were determined by qPCR using SYBR Green I Master mix (Roche Diagnostics, Basel, Switzerland) and the hexon primers (
9
Quantification of Adenoviral DNA
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Viral DNA was obtained from supernatants, cellular extracts or frozen tissues using the UltraClean BloodSpin DNA Isolation Kit (Mo Bio Laboratories, Carlsbad, CA) according to the manufacturer's instructions. Viral genomes were determined by Real-Time qPCR (100 ng of DNA) using SYBR Green I Master plus mix (Roche Diagnostics, Basel, Switzerland) and the hexon primer sequences: Fw 5′-GCCGCAGTGGTCTTACATGCACATC-3′ and Rv 5′-CAGCACGCCGCGGATGTCAAAG-3′. The adenovirus copy number was quantified with a standard curve, consisting of adenovirus DNA dilutions (102-107 copies) in a background of genomic DNA.
10
Adenoviral Transduction and Gemcitabine Sensitivity
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BxPC-3 and PANC-1 cells were infected with 4000 vp/cell and 200 vp/cell, respectively. Culture medium was removed at 4 h after infection and cells were treated, or not with 50 ng/ml and 500 μg/ml of gemcitabine (in BxPC-3 and PANC-1 respectively). Viral DNA was obtained from cell and supernatant (lysed by three freeze and thaw cycles) at 4 and 72 h after infection, using the UltraClean BloodSpin DNA Isolation Kit (Mo Bio Laboratories, CA USA) according to manufacturer’s instructions. Viral genomes were determined by Real Time PCR using SYBR Green I Master mix (Roche Diagnostics, Switzerland) and Hexon primers (described in Additional file 3 : Table S1). The adenovirus copy number was interpolated in a standard curve, consisting of adenoviral DNA dilutions in a background of genomic DNA.
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