Wizard genomic dna purification protocol

Manufactured by Promega

Sourced in United Kingdom

The Wizard® Genomic DNA Purification Protocol is a laboratory tool designed for the extraction and purification of genomic DNA from a variety of sample types. It utilizes a silica-based membrane technology to efficiently capture and isolate high-quality genomic DNA, which can then be used in various downstream applications.

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Lab products found in correlation

Lightcycler 480 probes master mix, by Roche (1 mentions) Nuclei lysis solution, by Promega (1 mentions) Sample purification beads, by Illumina (1 mentions) Trueseq nano dna library prep protocol, by Illumina (1 mentions) Phusion dna polymerase, by Thermo Fisher Scientific (1 mentions) Lysozyme, by Merck Group (1 mentions) Colistin sulphate, by Merck Group (1 mentions)

4 protocols using wizard genomic dna purification protocol

Quantitative PCR for Toxoplasma DNA Detection

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One cerebral hemisphere from each mouse was homogenised in 1 ml PBS using a syringe and 18G needle followed by a 21G needle. One lung from each mouse was homogenised in 1 ml Nuclei Lysis Solution (Promega Corporation, Southampton, UK) in Precellys tubes containing CK28 ceramic beads. Both eyes from each mouse were homogenised, together, in 1 ml Nuclei Lysis Solution using an 18G needle. DNA was extracted from 400 μl of each homogenised tissue using the Wizard® genomic DNA purification protocol (Promega Corporation, Southampton, UK) [21 (link)]. The final pellet of DNA was resuspended in 200 μl nuclease-free water and stored at -20 °C until required for PCR.
Quantitative PCR, targeting the 529 bp repeat element, was carried out in triplicate according to a previously described method [22 (link)] with slight modifications. The 20 μl reaction mixture consisted of 10 μl 2× Lightcycler® 480 Probes Master mix (Roche), 0.7 μM of each primer (Tox-9Fand Tox-11R), 0.1 μM of Tox-TP1, 0.2 μM of CIAC-probe, 0.02 fg of CIAC and 250 ng of template DNA in 8 μl. Sequences for primers and probes, as well as PCR conditions have been described previously [12 (link), 22 (link)].

Hamilton C.M., Black L., Oliveira S., Burrells A., Bartley P.M., Melo R.P., Chianini F., Palarea-Albaladejo J., Innes E.A., Kelly P.J, & Katzer F. (2019). Comparative virulence of Caribbean, Brazilian and European isolates of Toxoplasma gondii. Parasites & Vectors, 12, 104.

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Genomic DNA Isolation and CRISPR Profiling

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Genomic DNA was isolated from DG7710 cells by first treating with modified Wizard Genomic DNA Purification protocol for gram-positive bacteria (Promega): bacterial cell pellets were resuspended in 0.5 M EDTA, pH 8.0 supplemented with 200 μg ml⁻¹ lysozyme (Sigma) and incubated at 37 °C for 25 min prior to pelleting and removing the supernatant. The standard Wizard protocol for gram-positives was then followed as described by the manufacturer (Promega). We used 200 ng (log phase) of plasmid as input for PCR of the CRISPR locus with Phusion DNA Polymerase (Thermo) with the following primer mix: oPN737 with oPN738, oPN757, oPN758, or oPN759 depending on whether JAV25, JAV25-BIM01, JAV25-BIM02, or JAV25-BIM03 was used. To differentiate between samples during multiplexed high-throughput sequencing, variants of oPN737 containing randomized 5 nucleotides (NNNNN) followed by 3-6 nucleotide barcode at 5’ end. Amplicons corresponding to the size of expanded CRISPR arrays were gel purified allowing for the removal of unexpanded CRISPR arrays. Purified amplicons were then prepared for sequencing with the TrueSeq Nano DNA Library Prep protocol (Illumina), using a final concentration of 1.36x Sample Purification Beads (Ilumina) following end repair for further size selection, followed by high-throughput sequencing with the MiSeq platform.

Nussenzweig P.M., McGinn J, & Marraffini L.A. (2019). Cas9 cleavage of viral genomes generates immunological memories during the type II CRISPR-Cas response.. Cell host & microbe, 26(4), 515-526.e6.

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DNA Extraction and Toxoplasma Quantification

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DNA was extracted from 400 μl of digested tissue homogenate per chicken using the Wizard® genomic DNA purification protocol (Promega Corporation, Southampton, UK) [24 (link)]. The final pellet of DNA was resuspended in 200 μl molecular-grade water and stored at -80 °C prior to use. Extraction controls were included within each group of DNA extractions as described previously [21 (link)]. PCR amplifications, targeting the ITS1 region between the 18S and 5.8S rRNA genes, were carried out in duplicate using conditions previously described [25 (link)]. In order to quantify the level of T. gondii DNA present in the digested chicken tissues (used as the mouse inocula), any samples positive by ITS1 PCR were also screened using a quantitative PCR, targeting the 529-bp repeat element, as previously described [21 (link)].

Hamilton C.M., Kelly P.J., Boey K., Corey T.M., Huynh H., Metzler D., Villena I., Su C., Innes E.A, & Katzer F. (2017). Predominance of atypical genotypes of Toxoplasma gondii in free-roaming chickens in St. Kitts, West Indies. Parasites & Vectors, 10, 104.

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Transposon Mutant Library Generation and Colistin Selection

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The transposon mutant library in WT BAL062 was generated using an EZ:Tn5 transposon containing a kanamycin-resistance cassette (Epicentre Biotechnologies), as described previously [27 (link)]. The colony number was estimated and cells batch pooled as described in [28 (link)] to yield a total of 600 000 cells in the library. For the experiment, approximately 1×10⁹ cells were inoculated into 10 ml MH broth containing 0.05 mg colistin sulphate l⁻¹ (1/10th MIC; Sigma-Aldrich) and incubated overnight in a shaking incubator at 37 °C. The control did not contain colistin and experimental conditions were assessed in duplicate. The culture was serially passaged, taking 100 µl and inoculating 10 ml fresh broth with the same amount of colistin or none for the control. After an overnight incubation, DNA was extracted from 2 ml culture using the Wizard genomic DNA purification protocol (Promega) and sequenced as previously described [29 (link)].

Boinett C.J., Cain A.K., Hawkey J., Do Hoang N.T., Khanh N.N., Thanh D.P., Dordel J., Campbell J.I., Lan N.P., Mayho M., Langridge G.C., Hadfield J., Chau N.V., Thwaites G.E., Parkhill J., Thomson N.R., Holt K.E, & Baker S. (2019). Clinical and laboratory-induced colistin-resistance mechanisms in Acinetobacter baumannii. Microbial Genomics, 5(2), e000246.

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