Notes: Fungi of the genus Cercospora are plant pathogens that cause leaf spot and blight diseases, and produce the polyketide toxin cercosporin. The bacterium Xanthomonas campestris is able to rapidly degrade cercosporin. In this study, X. campestris mutants unable to degrade cercosporin were created by chemical mutagenesis. Complementation studies with a plasmid-based library of X. campestris DNA showed that the ability to degrade cercosporin was restored upon transformation with plasmids containing an oxidoreductase gene and a putative transcriptional regulator. These genes were then amplified from the mutant strains by high-fidelity PCR. The PCR products were separated by agarose gel electrophoresis, purified using the Wizard^® SV Gel and PCR Clean-Up System, and subcloned into the pGEM^®-T Easy Vector. The mutant genes were then sequenced to identify the nature of the mutations. (3531)

Notes: In the process of demonstrating the role of IN in HIV-1 integration in yeast, the authors purified all DNA vectors and PCR products with the Wizard® Plus SV Miniprep System and Wizard® SV Gel System. PCR products were generated using Taq DNA Polymerase. The pGEM®-T Vector was used to clone amplification products. Sequencing was performed using BamHI, religated with T4 DNA Ligase. (3704)

Notes: In this study, insertional mutagenesis with the transposon TN5367 was used to generate a library of M. paratuberculosis mutants. Sequences containing transposons were then amplified, gel purified using the Wizard^® SV Gel and PCR Clean-Up System, and cloned into the pGEM^®-T Easy Vector prior to sequencing. Bioinformatic screening was then used to identify potential virulence determinants for further study in a mouse model of M. paratuberculosis infection. (3534)

Notes: A deletion mutation of the serine/threonine protein kinase NDR2 was created by PCR using two mutagenesis primers and two plasmid-based primers. After amplification, the two products were run on a 1% agarose gel and extracted using the Wizard® SV Gel and PCR Clean-Up System. The purified fragments were mixed, annealed, re-amplified and then digested prior to cloning into an expression vector. The human colorectal cancer cell lines HCT-116, DLD-1, and SW480 used in the study were seeded into a 24-well plate at 5 × 10⁴/well, and transfected using the TransFast™ Transfection Reagent. The transfection was assessed with a green fluorescent protein expression vector. (3438)

Notes: These researchers used laser-capture microscopy followed by real-time PCR to detect and identify Cryptosporidium oocysts in stained fecal smears and water samples on glass slides. After microdissection of single oocysts or groups of oocysts from the stained slides, DNA was extracted and real-time PCR performed using primers specific for the cryptosporidial 18s rRNA gene. To confirm primer specificity and the identity of the real-time PCR products, the amplimers were recovered from the LightCycler® capillaries at the end of each real-time experiment. Products were then separated on agarose gels and purified using the Wizard® SV Gel and PCR System prior to sequencing using a BigDye® terminator cycle sequencing kit from Applied Biosystems. (3532)

Notes: To examine if the depletion of Drosophila transcription termination factor (DmTTF) after RNAi treatment could reduce the gene copy number, genomic DNA was isolated from RNAi-treated and untreated Drosophila embryonic D.Mel-2 cells using the Wizard^® Genomic DNA Purification Kit. The mitochondrial ND3 gene and the nuclear H2B histone gene were used as probes for the Xho I-digested, Southern-blotted genomic DNA to compare the treatment groups. The Wizard^® SV Gel and PCR Clean-Up System was used to clean up the PCR-amplified probes. (3418)

Notes: Production of extracellular proteins in E. Carotovora subspecies that are critical for the development of soft-rotting disease of plants, is controlled by quorum-sensing signals, plant signals and assorted transcriptional factors and post-transcriptional regulators. Of these, post-transcriptional regulation by the RsmA-RsmB RNA pair is critical. RsmA is a small RNA-binding protein that promotes decay of RNA. RsmB specifies an untranslated regulatory RNA that binds RsmA and neutralizes its regulatory effect. Many of the transcription factors and QS signals known to regulate extracellular protein production actually act via these post-transcriptional regulators. ExpR, the putative AHL (N-acetyl homoserine lactone) receptor of E. carotovora subspecies carotovora, activates transcription of rmsA and AHL prevents this activation. The authors generated PCR fragments using primers for rsmA71 and rsmA153 and then used the Wizard(R) SV Gel and PCR Clean-Up System (Cat.# A9281) to purify PCR-generated DNA fragments used in gel mobility shift assays. Band shift assays revealed that ExpR is a DNA-binding protein and that its DNA-binding property is modified by AHL. In addition they showed that RsmA overproduction is responsible for inhibition of extracellular protein. (3572)

Notes: Researchers used the Wizard^® Genomic DNA Purification Kit to isolate genomic DNA from non-Agrobacterium field bacteria samples. The isolated genomic DNA was used in PCR to amplify regions of the 16S rRNA gene. The PCR products were cleaned up using the Wizard^® SV Gel and PCR Clean-Up System and used in sequencing reactions. The paper also mentions the use of the Wizard^® Magnetic DNA Purification System for Food to isolate Agrobacterium radiobacter from cucumber root mats grown in the lab. (3190)

Notes: Researchers used GoTaq^® DNA polymerase to amplify 139bp and 815bp regions of hOST-PTP cDNA for detection and probe synthesis. The full-length 4006bp cDNA was amplified with Pfu DNA Polymerase. Fragments were gel purified using the Wizard^® SV Gel and PCR Clean-Up System prior to cloning into the pGEM^®-T Easy Vector.  The Prime-a-Gene^® Labeling System was used to make ³²P-dCTP labeled probes, which were used to screen cDNA clones. (3111)

Notes: The Wizard® SV Gel and PCR Clean-Up System was used to purify a mutant ~870bp prostaglandin E synthase (PGES) PCR product. The purified fragment was then subcloned into the pLenti6/V4-D-TOPO vector and sequenced. (2749)

Notes: Researchers used the Wizard^® SV Gel & PCR Clean-Up System to purify rat c-fos RT-PCR products that were then quantified by A₂₆₀ absorbance. These products were used as standards for quantitative PCR reactions. The researchers used an Applied Biosystems Prism 7000 sequence detection system to perform the 50μl quantitative PCR reactions. (2678)

Notes: The Wizard^® SV Gel and PCR Clean-Up System was used to purify a 280 base-pair PCR product, which was then directly sequenced. (2791)