Transposon insertion sites of CDI-resistant mutants were determined using arbitrary PCR, as described previously (15 (link)). Genomic DNA was extracted from transposon mutants and wild-type AU0158 strain (as a control) using the Wizard Genomic DNA Purification system (Promega) according to the manufacturer’s protocol. Nested arbitrary-primed PCR was performed using this genomic DNA as template with primers Arb1 (arbitrary primer) and Tn3out (first round primer annealing to the 3′ end of the transposon) (Table S4). PCR products were treated with ExoSAP-IT PCR Product Cleanup Reagent (Applied Biosystems) or ExoProStar (Cytiva) and used as templates for the second, nested PCR with primers Arb2 and Tn3in. Second-round PCR products from transposon mutants were compared to the wild-type AU0158 negative control by agarose gel electrophoresis and treated with ExoSAP-IT or ExoProStar. The transposon-chromosome junctions in the second-round PCR products were sequenced with primer Tn3seq and transposon-disrupted genes identified by BLAST analysis.
Wizard genomic dna purification system
Manufactured by Promega
Sourced in United States, United Kingdom
The Wizard Genomic DNA Purification System is a kit designed for the isolation and purification of genomic DNA from a variety of sample types, including human, animal, plant, and microbial cells. The system utilizes a simple, fast, and efficient method to extract high-quality genomic DNA that is suitable for downstream applications such as PCR, restriction enzyme analysis, and sequencing.
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Lab products found in correlation
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Specific oligonucleotide primers, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Exosap it pcr product cleanup reagent, by Thermo Fisher Scientific (1 mentions)
Ez dna methylation lightning kit, by Zymo Research (1 mentions)
Infinium humanmethylation450 beadchip array, by Illumina (1 mentions)
Taqman allelic discrimination assay, by Thermo Fisher Scientific (1 mentions)
Cfx96 real time polymerase chain reaction detection system, by Bio-Rad (1 mentions)
Quantifluor one dsdna system, by Promega (1 mentions)
Nextera xt library prep kit, by Illumina (1 mentions)
Quant it picogreen, by Thermo Fisher Scientific (1 mentions)
Veriti 96 well thermocycler, by Thermo Fisher Scientific (1 mentions)
Gelred, by Biotium (1 mentions)
Agilent 2200 tapestation system, by Agilent Technologies (1 mentions)
Qubit dsdna br assay kit, by Thermo Fisher Scientific (1 mentions)
Plant fungi total rna purification kit, by Norgen Biotek (1 mentions)
Methylflash methylated dna quantification kit, by Epigentek (1 mentions)
Taq dna polymerase, by Thermo Fisher Scientific (1 mentions)
Thermal cycler, by Bio-Rad (1 mentions)
20 protocols using wizard genomic dna purification system
1
Mapping Transposon Insertion Sites in CDI-resistant Mutants
2
Methylation Profiling of ccRCC
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Twenty-four matched DNA samples (12 ccRCC and 12 matched normal-surrounding kidney tissues) were used for DNA methylation profiling. Twelve ccRCC tissue samples were randomly allocated with pathologic stage. Genomic DNA (gDNA) was extracted by standard methods by using the Wizard Genomic DNA Purification System (Promega, Madison, WI, USA). Bisulfite-modified gDNA was prepared using EZ DNA Methylation-Lightning kit (Zymo Research, Orange, CA, USA) according to the manufacturer's instructions. The methylation status was assayed using the Infinium HumanMethylation450 BeadChip array (Infinium Methylation 450K; Illumina Inc., San Diego, CA, USA), which enables interrogation of the methylation status of more than 480,000 CpG sites distributed over the whole genome. Fluorescence signals corresponding to C- or T-nucleotides were measured, and the data were used to assign a quantitative measure of methylation level of specific CpG islands.
3
Genotyping of Pharmacogenomic Variants
4
Complete Genome Sequencing of Isolated Bacterium
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The selected isolated bacterium (strain 21p) was incubated in 40 mL of liquid tryptic soy broth 10% for 48 h, 30 °C and 400 rpm (SK-O330). The media was centrifuged until 1 mL of bacteria pellet was obtained. DNA was extracted with Wizard® Genomic DNA Purification System (Promega, Leiden, The Netherlands) using the manufacturer’s protocol. DNA integrity was confirmed in the electrophoreses gel. DNA quantity and quality were assessed by fluorometry using a QuantiFluor® ONE dsDNA System (Promega Corporation, Fitchburg, WI, USA) and ratios 260/280 and 260/230 absorbance by spectrophotometry.
The genome was sequenced using Illumina, for high quality short reads sequencing and MinION, for long reads to achieve a complete genomic sequence. For Illumina, the genomic DNA library was constructed using a Nextera XT library prep kit (Illumina, Inc., San Diego, CA, USA) with paired-end reads (2 × 150 bp) on a MiSeq v3 platform 1GBps (Illumina, San Diego, CA, USA). For MiniON sequencing, libraries were prepared with Rapid Barcoding Sequencing QK-RBK004 (Oxford Nanopore Technologies [ONT], Oxford, UK) platform using the MinKNOW software, Version 4.5.0 (Figure S1 ).
The genome was sequenced using Illumina, for high quality short reads sequencing and MinION, for long reads to achieve a complete genomic sequence. For Illumina, the genomic DNA library was constructed using a Nextera XT library prep kit (Illumina, Inc., San Diego, CA, USA) with paired-end reads (2 × 150 bp) on a MiSeq v3 platform 1GBps (Illumina, San Diego, CA, USA). For MiniON sequencing, libraries were prepared with Rapid Barcoding Sequencing QK-RBK004 (Oxford Nanopore Technologies [ONT], Oxford, UK) platform using the MinKNOW software, Version 4.5.0 (
5
Genome Assembly from 454 Sequencing
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Genomic DNA from the TFA strain was prepared by using the Wizard Genomic DNA purification system (Promega). Its quality and quantity was assessed with Quant-It-Picogreen (Invitrogen) and a Nanodrop ND-1000 Spectrophotometer (Thermo Fisher Scientific). 12.5 μg of purified DNA was sent to the company LifeSequencing (http://www.lifesequencing.com/ , Valencia) for sequencing using a 454 GS-FLX platform. Assembly of the raw sequences by a Celera assembler resulted in 42 contigs. Final assembly was done by searching each contig end against the UniProt database using BLASTx [42 (link)], detecting truncated proteins (bridge proteins), which match in more than one contig end and designing primers at the end of those contigs to fill the gaps [43 (link)]. Some additional steps were needed to close the genome, consisting of Southern Blot assays against a TFA genomic DNA library using probes designed from contig ends, and then sequencing positive cosmids in order to find contiguous ends.
6
Genomic DNA Extraction and Genotyping
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DNA extraction was performed with the ‘Wizard’ Genomic DNA purification system according to the manufacturer's instructions (Promega, Southampton, UK), using mouse tail tips taken before the perfusion. Specific oligonucleotide primers (Invitrogen, Loughborough, UK) were used for genotyping against erk1, erk2, synapsin‐cre, GFAP‐cre.
7
Genomic DNA Isolation and Duplex-PCR for nifH and nodC
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The bacterial isolates were grown in liquid YM medium for three days for the fast-growing isolates and six days for the slow-growing. An aliquot of 1 mL of each broth was used for the DNA extraction using the Wizard® Genomic DNA Purification System (Promega, USA) according to the manufacturer's instructions. nifH and nodC genes were co-amplified in a duplex-PCR reaction as described by Fernandes Júnior et al.24 For the nifH, the primers PolF (TGCGAYCCSAARGCBGACTC) and PolR (ATSGCCATCATYTCRCCGGA)25 (link) were used. For nodC, the primers NodCF (AYGTHGTYGAYGACGGTTC) and NodCR(I) (CGYGACAGCCANTCKCTATTG)26 (link) were applied. For a single isolate that showed positive amplification of the nifH amplicon, a complementary uniplex-PCR was performed with the primers nodCForB (CTCAATGTACACARNGCRTA) and nodCRevB (GAYATGGARTAYTGGYT)57 (link) targeting the nodC amplification of β-rhizobia.
The reactions were performed in a Veriti 96-well thermocycler (Applied Biosystems, USA) and the PCR products were submitted to horizontal electrophoresis in agarose gel (0.8%, w/v). The gel was stained with Gel Red (Biotium) and visualized in a UV chamber. The bacterial isolates positive for both amplicons were selected for the next steps.
The reactions were performed in a Veriti 96-well thermocycler (Applied Biosystems, USA) and the PCR products were submitted to horizontal electrophoresis in agarose gel (0.8%, w/v). The gel was stained with Gel Red (Biotium) and visualized in a UV chamber. The bacterial isolates positive for both amplicons were selected for the next steps.
8
Genomic DNA Isolation from Trichuris Worms
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Adult worms dwelling in the large intestine in sheep were collected from naturally infected sheep from a private farmer in Luotian, Hubei, PR China. The worms were extensively washed with physiological saline and identified initially as Trichuris spp. based on the morphological characteristics and predilection site [11 (link),25 (link)]. The worms were then fixed in 70% ethyl alcohol and stored at −20 °C until further used for experiments. Standard sodium dodecyl/proteinase K treatment was done followed by mini column purification method (Wizard Genomic DNA Purification System, Promega, Beijing, China) to isolate the genomic DNA.
9
Leishmania infantum DNA Extraction
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Promastigotes from positive cultures were expanded at 25°C in Schneider’s insect medium, pH 7.2, supplemented with 10% (v/v) FBS and 2% (v/v) male human urine. The parasites were grown to a density of 1×109 cells/mL (late log phase) and washed twice with PBS (pH 7.2) before DNA extraction.
Total DNA was extracted from spleen and from L. infantum promastigotes kept in vitro. DNA extraction was carried out using the Wizard Genomic DNA Purification System (Promega, Madison, WI, USA) following the manufacturer's instructions, which included a prior digestion phase with 17.5 μL of proteinase K (20 mg/mL) for 12 h at 55°C. The DNA was dissolved in 100 μL of tris EDTA buffer (TE buffer).
Total DNA was extracted from spleen and from L. infantum promastigotes kept in vitro. DNA extraction was carried out using the Wizard Genomic DNA Purification System (Promega, Madison, WI, USA) following the manufacturer's instructions, which included a prior digestion phase with 17.5 μL of proteinase K (20 mg/mL) for 12 h at 55°C. The DNA was dissolved in 100 μL of tris EDTA buffer (TE buffer).
10
Mycelial DNA and RNA Extraction Protocols
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For the DNA extraction, mycelial samples were harvested and freeze dried for 24 h. The total DNA was isolated from the freeze-dried samples using the Wizard® Genomic DNA Purification System (Promega, Madison, WI, USA) as per the manufacturer’s instructions to achieve high-molecular-weight DNA. The eluted DNA was quantified using a QubitTM dsDNA BR Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA), and the quality was assessed using a Nanodrop 2000 (Thermo Fisher Scientific). The integrity and molecular weight were assessed using an Agilent TapeStation 2200 system (Agilent Technologies, Santa Clara, CA, USA) following manufacturer’s instructions.
For RNA extraction, mycelial samples were collected into 1.5 mL Eppendorf tubes and stored at −80 °C until they were processed. The RNA extraction was carried out using a Plant/Fungi Total RNA Purification Kit (Norgen Biotek Corp., Thorold, ON, Canada). Quality was assessed using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific), and integrity was assessed using an Agilent TapeStation 2200 system.
For RNA extraction, mycelial samples were collected into 1.5 mL Eppendorf tubes and stored at −80 °C until they were processed. The RNA extraction was carried out using a Plant/Fungi Total RNA Purification Kit (Norgen Biotek Corp., Thorold, ON, Canada). Quality was assessed using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific), and integrity was assessed using an Agilent TapeStation 2200 system.
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