The length of the modM 5′-(CAAC)n-3′ tetranucleotide repeat tract in M. catarrhalis 195ME, and the proportion of each fragment length present was determined using GeneScan fragment analysis as per [23 (link)]. Briefly, the modM repeat region was amplified with the 6-carboxyfluorescein (6-FAM) labelled forward primer Mcmod2F-6FAM (5′-[6FAM]-TTACTTGACACTCTGAATGGA-3′) and the unlabelled reverse primer McmodrepR2 (5′-GTATTATGGGCAGTTTTTAAG-3′) using GoTaq Flexi DNA polymerase (Promega) in 20 μL reaction volumes as per manufacturer’s instructions. Analysis of fluorescent traces and quantification of fragment lengths was performed using PeakScanner v1.0 (Applied Biosystems, Grand Island, NY, USA). M. catarrhalis subpopulations containing different modM3 repeat tract lengths were selected by fragment length analysis of individual colonies, as previously described [20 (link)].
Gotaq flexi dna
Manufactured by Promega
Sourced in United States
GoTaq Flexi DNA is a thermostable DNA polymerase enzyme designed for use in the polymerase chain reaction (PCR) process. It enables the amplification of DNA sequences from a variety of sample types. The enzyme provides flexibility in PCR optimization and is suitable for a wide range of applications.
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Lab products found in correlation
5 protocols using gotaq flexi dna
1
Determination of modM Tetranucleotide Repeats in Moraxella catarrhalis
2
Improved PCR Protocol for Genomic DNA Amplification
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The PCR were done as described by Díaz et al. (2012 (link)) with a few modifications. The PCR was carried out in 20 µl reactions comprising of 2.5 µl of 20 ng/µl genomic DNA dissolved in 1× TE buffer, 0.4 µl of 10 mM dNTPs (Promega UK LTD) dissolved in 1× TE buffer, 1.6 µl of 25 mM MgCl2, 4.0 µl of 5× clear buffer, 1 µl each of 5 µM (dissolved in 1× TE buffer) forward and reverse primers, 0.080 µl GO TAQ FLEXI DNA (Promega UK LTD) polymerase (5U/µl), 9.42 µl of double distilled water.
3
PCR Screening for Recombination Events
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Example 5
PGR was performed using GoTaq Flexi DNA polymerase (Promega) with primers HOP′ attH4X_F1 (SEQ ID NO: 25) and PuroRev24 (SEQ ID NO: 26) and 200 ng of genomic DNA as template per PCR reaction in 50 μl volume. The following thermal cycling parameters were used for the PCR: an initial step of 95° C. for 5 minutes, 35 cycles of 95° C. for 1 minute, 57° C. for 30 seconds and 72° C. for 1 minute, and a final step of 72° C. for 5 minutes. The PCR samples were analyzed by electrophoresis in 0.8% agarose gel in Tris-Boric acid-EDTA buffer.
4
Plasmid DNA Isolation and Amplification
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DNA cloning and gel electrophoresis were performed according to standard procedures [21 ]. Plasmid DNA was isolated using the Plasmid Mini Isolation Kit or the Illustra Plasmid Prep Mini Spin Kit (GE life science, US) according to the manufacturer’s instructions. All restriction enzymes, ligase, and DNA ladder were purchased from New England BioLabs (US) and used according to the manufacturer’s instructions. The amplification of DNA was conducted on a Veriti Thermal Cycler (Applied Biosystems, US) using GoTaq Flexi DNA (Promega, US) using standard conditions. The primers used in this study were synthesized by the Molecular Biotechnology Company (Brazil). PCR products were separated on agarose gels and purified using an Illustra GFX PCR DNA and Gel Band Purification (GE Healthcare, US) according to the manufacturer's instructions.
5
Microsatellite Profiling of PKP2 Locus
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To establish whether the rearrangements involving all the PKP2 genomic sequence observed in 5 apparently unrelated subjects could be ascribed to the same mutational event or arose independently, we evaluated 7 microsatellite markers spanning a region of ≈2.3 Mb across the PKP2 locus. In addition, these markers were used for genotyping an additional subject carrying a 122-kb deletion in chromosome 12 encompassing the entire PKP2 gene who came to our attention because of this deletion but without any clinical signs of the disease. Genetic markers' primers sequences were obtained from the UCSC Genome Browser website (http://genome. ucsc.edu/). Markers were PCR amplified from genomic DNA using GoTaq Flexi DNA polymerase (Promega) in a 12.5 µL reaction volume, separated on an ABI3500 Dx, and analyzed with GeneMapper v. 3.1 software (Applied Biosystems).
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