Notes: 0.5 U of GoTaq^® Flexi DNA polymerase was used to evaluate loci for polymorphism across a sample of 30 individual plants. (4892)

Notes: The authors of this study used real time-qPCR and PCR-DGGE (denaturing gradient gel electrophoresis) to analyze and compare the mixed bacterial systems from fecal samples of vegetarians and omnivores. Bacterial DNA was isolated from frozen fecal samples using the Maxwell® 16 Tissue DNA Purification Kit, and PCR-DGGE reactions were set up using GoTaq® Flexi DNA Polymerase and GoTaq® Flexi Colorless Reaction Buffer. The authors of this study were able to detect differences in microbiota that seemed to be related to diet. (4523)

Notes: The authors were interested in quantitating the presence as well as the prevalence of Lactobacillus gasseri K7 in humans that did not consume the probiotic bacteria. Fecal samples from 45 healthy adults were collected, frozen, diluted, centrifuged and digested with proteases. After sonication, DNA was extracted using the Maxwell® 16 Tissue DNA Purification kit on the Maxwell® 16 instrument. This same kit and instrument also were used to isolate bacterial DNA from 1ml bacterial cultures. The purified DNA was PCR amplified using primers for gassericin K7 A and K7 B (bacteriocin) genes and GoTaq® Flexi DNA Polymerase in Green GoTaq® Flexi Buffer for 30 cycles. PCR products were analyzed by 1.8 % agarose gel electrophoresis. (4522)

Notes: Human Genomic DNA used as the starting material in the NGS workflow.  GoTaq® Flexi Colorless Buffer and GoTaq® Flexi Polymerase were used in amplification of template in step added to the beginning of library preparation. (4531)

Notes: These authors investigated the differences in gut microbial flora between obese and normal-weight subjects. They used the Wizard® Genomic DNA Purification Kit to extract DNA from diluted fecal extracts. The extracted DNA was analyzed by PCR to identify Bacteroidetes and Firmicutes. Differences in distribution of these phyla was between the subject groups were identified. (4220)

Notes: The authors investigated the suppression of E. coli O157:H7 in sand-based livestock bedding and hypothesized that suppression of E. coli O157:H7 growth was mediated by an environmentally stable population of pathogen-suppressing bacteria. These bacteria were identified by terminal restriction fragment length polymorphism (T-RFLP) analysis of amplified 16S rRNA gene sequences isolated from used bedding followed by cloning and sequencing of the most abundant terminal restriction fragments. Amplifications were performed using the GoTaq^® Flexi DNA Polymerase, then PCR products were cloned into the pGEM^®-T Easy Vector. The PureYield™ Plasmid Miniprep System was used to purify plasmids for sequencing. (4165)

Notes: Sequence variation within a gene, such as single nucleotide polymorphisms (SNPs), can lead to differences in expressions levels of corresponding mRNAs; genes that are self-regulated by this mechanism (cis modulation) are difficult to identify accurately with existing techniques. The authors used bioinformatic and molecular approaches to estimate error rates when identifying cis-modulated transcripts and developed a simple method to detect these transcripts in C57BL/6J F^₁ hybrid mice. This method, which they named allelic specific expression, is RT-PCR-based and uses PCR primers that flank an informative SNP to quantify the differential expression levels of transcripts. GoTaq^® Flexi DNA Polymerase was used in the PCR. (4051)

Notes: The vasculoproliferative disorder peliosis hepatis has been linked to Bartonella henselae infection in humans and dogs. The authors sought to determine if this is true in cats, the natural reservoir for B. henselae, using immunohistochemistry (IHC) and PCR. Tissue sections from 26 cats with peliosis hepatis were formalin-fixed and paraffin-embedded and subjected to IHC using a B. henselae-specific antibody. To detect B. henselae DNA, PCR was performed using GoTaq® Flexi DNA Polymerase, genus-specific primers for the pap31 gene of Bartonella, 1mM MgCl₂ and total DNA isolated from 8µm paraffin-embedded tissue sections. The presence of Bartonella DNA was confirmed by PCR using species-specific primers that target the B. henselae heat-shock protein (htrA). These studies found no link between B. henselae infection and peliosis heptis in cats. (4094)

Notes: The authors describe a new form of PCR, co-amplification at lower denaturation temperature PCR (COLD-PCR), to detect low-level somatic mutations. This technique is based on the facts that a) each DNA sequence has a critical denaturation temperature (T_c), which is lower than the melting temperature (T_m) and below which PCR efficiency decreases dramatically and b) Tc depends on DNA sequence. The authors used GoTaq^® Flexi DNA Polymerase and mutation-specific TaqMan^® probes for tumor protein 53 (TP53) and epidermal growth factor receptor (EGFR) to detect low-level somatic mutations in a mixture of wildtype and mutant DNAs. Conventional TaqMan^_® technology can detect mutant alleles at an abundance of 10–20% of that of the wildtype allele; using COLD-PCR the authors were able to increase selectivity 15- to 30-fold, detecting as little as 0.8% mutuant alleles. (4038)

Notes: The authors characterized differential expression of several carcinogen metabolism genes in human alveolar compartment (AC) and bronchial epithelial compartment (BEC) lung tissues in smokers, former smokers and people who have never smoked. They combined laser capture microdissection (LCM) and quantitative RT-PCR. RNA was isolated from paired microdissected malignant and nonmalignant lung tissue, 100ng of total RNA was reverse transcribed in a 20µl reaction, then 1µl of cDNA was amplified by real-time PCR using an ABI PRISM® 7500HT sequence detection system, GoTaq® Flexi DNA Polymerase and gene-specific primers. The expression level for each gene was normalized using glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The results showed that expression of cytochrome P450 1B1 and glutathione-S-transferase P1 in AC, but not BEC, tissue was strongly associated with exposure to tobacco. (4095)

Notes: To determine whether oocytes are able to select X-bearing or Y-bearing spermatozoa, the authors performed in vitro fertilization of bovine oocytes with X-sorted semen, Y-sorted semen, a mixture of X- and Y-sorted semen, and unsorted semen. The gender of the resulting embryos was determined by amplifying two DNA targets: a Y chromosome-specific target for gender assignment and a bovine-specific satellite sequence as a control. PCRs were performed using GoTaq^® Flexi DNA Polymerase (1 unit per 25µl reaction), and amplified products were analyzed by agarose gel electrophoresis followed by ethidium bromide staining. (3881)

Notes: To determine the usefulness of multilocus sequence typing (MLST) in differentiating Candida species during epidemiological studies, the authors investigated the population structure of C. dubliniensis by amplifying the same 10 MLST loci found to be useful in differentiating isolates of C. albicans, a closely related species. PCRs were performed using 1.25 units of GoTaq^® Flexi DNA Polymerase and 1ng of DNA template in a 50µl reaction. (3880)

Notes: The authors knocked down expression of the hydin gene, which encodes a flagellar protein, to determine the function and location of hydin in Chlamydomonas reinhardtii. Amplification steps in the creation of the RNAi expression vector to target hydin expression were performed using GoTaq^® Flexi DNA Polymerase. (3723)

Notes: The authors studied hsp70 mRNA degradation in Drosophila Schneider cells. mRNA deadenylation and decay were monitored by Northern blot. Two of the Northern blot probes used to visualize the mRNA decay products were synthesized by transcription of linearized plasmids using T7 RNA Polymerase and [α-³²P] UTP. A population of deadenylated mRNA was created by hybridizing mRNA with oligo(dT) and treating with RNase H. CCR4•NOT was identified as the main deadenylase involved in mRNA decay, and the PAN2:PAN3 deadenylase was a minor contributor. RNA interference was used to knock down expression of PAN2 and CAF1, a subunit of CCR4•NOT, to assess the effect on mRNA decay. Reduced expression levels of PAN2 and CAF1 were confirmed by semi-quantitative RT-PCR and Western blotting, respectively. RT-PCR was performed using 1.5µg total RNA and 150 units of MMLV Reverse Transcriptase in a 25µl reaction. One microliter of the RT reaction was used as a template in an 80µl PCR using 0.5 units of GoTaq^® DNA Polymerase, 1.5mM MgCl₂ and 1X Green GoTaq^® Flexi Reaction Buffer. (3707)

Notes: The authors examined whether genetic differences at the HLA class I locus affect development of Epstein Barr Virus-associated diseases. Peripheral blood mononuclear cells were isolated from asymptomatic EBV-seropositive and seronegative individuals and patients with acute infectious mononucleosis. DNA was isolated, and genotypes at two HLA class I loci and one HLA class III locus, as a control, were determined by PCR. The 10µl PCRs contained 25ng of DNA, 1X GoTaq^® Flexi Reaction Buffer, 2.5mM MgCl₂, 200µM dNTP, 0.5 units of GoTaq^® Flexi DNA Polymerase and 25µM of forward and reverse primer, one of which was labeled with 6-FAM fluorescent dye. The results show that HLA class I polymorphisms might predispose people to develop infectious mononucleosis upon EBV infection. (3712)

Notes: These authors used chromatin immunoprecipitation to show that neuron-restrictive silencer factor interacts with the ubiquitin carboxy-terminal hydrolase L1 (UCHL1) promoter in U87-MG, DMS53 and HeLa cells. The neuron-restrictive silencing element (NRSE1) of the UCHL1 promoter was amplified using GoTaq^® Flexi DNA Polymerase in a 25µl PCR. (3705)