ANI was calculated as an alternative to DNA–DNA hybridization according to a previous study (42 (link)). Whole genomic DNA from strain SCS5 was extracted using the Wizard SV Genomic DNA kit (Promega, USA). Sequencing was performed using an Illumina HiSeq 2500 instrument with a paired-end library (300 bp). Genome sequences were assembled in silico using SOAPdenovo2 (32 (link)). An ANI analysis was performed using the software JSpecies v1.2.1 (42 (link)). The DNA G+C content of strain SCS5 was determined by calculating the total amount of G and C bases from the whole genome sequence as a percentage.
Wizard sv genomic dna kit
Manufactured by Promega
Sourced in United States
The Wizard SV Genomic DNA Purification System is a kit designed for the rapid isolation and purification of high-quality genomic DNA from a variety of sample types, including animal cells, tissues, and blood. The kit utilizes a silica-based membrane technology to efficiently capture and purify genomic DNA, which can then be eluted in a small volume of buffer for downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Hiseq 2500, by Illumina (1 mentions)
Sybr green system, by Thermo Fisher Scientific (1 mentions)
Steponeplus pcr system, by Thermo Fisher Scientific (1 mentions)
Ex taq dna polymerase, by Takara Bio (1 mentions)
Tgradient thermocycler, by Analytik Jena (1 mentions)
Pyes2, by Thermo Fisher Scientific (1 mentions)
Qiaspin miniprep kit, by Qiagen (1 mentions)
Accuoligo, by Bioneer (1 mentions)
Pegfp c1, by Takara Bio (1 mentions)
Qiaquick pcr purification kit, by Qiagen (1 mentions)
4 protocols using wizard sv genomic dna kit
1
Genome Sequencing and ANI Analysis
2
Quantifying Brain Parasite Burden in Mice
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Brain parasite burden in mice was determined by quantitative real-time PCR (qRT-PCR), as previously described (Wahab et al., 2010 (link)). The primers were engineered to detect the repetitive area of 529 bp to T. gondii, using SYBR green system (Invitrogen, San Francisco, CA). The DNA extraction (from the right cerebral hemisphere) was carried out using Wizard SV Genomic DNA kit (Promega Co., Madison, WI), by taking macerated brain sample (20 μg) following manufacturer's instructions. The total DNA concentration was detected by UV spectrophotometry (260 nm) adjusting the concentration of DNA samples to 200 ng/μl. The assays to determine the T. gondii quantification were performed using “StepOnePlus™ PCR Systems” (Applied Biosystems, Foster City, CA), with Master Mix (Promega Co., Madison, WI), with 900 nM of each primer. All reactions were conducted simultaneously with the positive control samples carrying out a standard curve following seven 10-fold dilutions, starting from 1:10 containing 100 ng of parasite DNA. The parasite quantifications were counted by interpolation from a standard curve (10−2 a 10−7 ng), with equivalent of T. gondii DNA.
3
Genotyping Transgenic Mice for Myc Expression
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Genomic DNA was isolated from mouse tail clipping using a Wizard SV genomic DNA kit (Promega). qPCR was performed for LANA and ApoB primers as previously described [28 (link)]. Mice with overexpressed Myc were typed by PCR according to supplier’s protocol using primers oIMR8447 & oIMR8448 for wild-type mice and oIMR8450 & oIMR8453 for the Myc mice (http://jaxmice.jax.org/protocolsdb/f?p=116:2:3011848657952163::NO:2:P2_MASTER_PROTOCOL_ID,P2_JRS_CODE:5234,008341 ).
4
Genomic DNA and Plasmid Isolation Protocol
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Chromosomal DNA was prepared from S. cerevisiae s2805 using a Wizard SV genomic DNA kit (Promega, Madison, WI, USA). Plasmid DNA such as pYES2.0 (Invitrogen, Carlsbad, CA, USA) and pEGFP-C1 (Clontech Laboratories, Palo Alto, CA, USA) were prepared from Escherichia coli cells using an alkaline lysis technique with a QIA spin Miniprep kit (Qiagen, Hilden, Germany). DNA modification, analysis by agarose gel electrophoresis, and ligation were performed using standard procedures [10 ]. The PCR experiments were carried out using a T Gradient thermocycler (Biometra, Göttingen, Germany), Ex Taq™ DNA polymerase (Takara Bio, Shiga, Japan), and chromosomal DNA as the template. The PCR products were purified using a QIA quick PCR purification kit (Qiagen). The oligonucleotides used for PCR amplification were purchased from Accu Oligo (Bioneer, Daejeon, Korea). Table 1 lists the primers used to introduce the base pair changes. The transformation of E. coli was performed by electroporation with an Electro Cell Manipulator (BTX Technologies, Hawthorne, NY, USA) and yeast transformation was performed by lithium acetate method [11 (link)].
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