DNA was extracted from manual microdissected sections of FFPE tissue focussed on areas with high tumour cell percentage by incubation in 5% Chelex‐100 in TET lysis buffer and 10% Proteinase K (20 mg/ml) (Qiagen, Hilden, Germany) for 16 h at 56°C. MSI analysis was performed using five mononucleotide repeat markers (NR‐21, NR‐24, NR‐27, BAT‐25, and BAT‐26) in a single multiplex PCR.23 The PCR was carried out on a MJ Research PTC‐200 Thermal Cycler using 5PRIME HotMaster Taq DNA polymerase (QuantaBio, Beverly, MA, USA) with 1 μl DNA and the following program: initial denaturation at 94°C for 2 min, 35 cycles of denaturation at 94°C for 20 s, annealing at 55°C for 10 s, and extension at 65°C for 30 s, with a final extension at 65°C for 7 min. DNA fragment analysis was executed on the 3730 DNA Analyser (Applied Biosystems, Foster City, CA, USA). Product sizes for the markers were determined using GeneMarker V.2.6.7 (Applied Biosystems). Normal colon tissues were used as control. A tumour was defined as MSI if at least two of the five markers showed instability.
5prime hotmaster taq dna polymerase
Manufactured by Quantabio
Sourced in United States
The 5PRIME HotMaster Taq DNA polymerase is a thermostable DNA polymerase enzyme used for DNA amplification in molecular biology applications. It exhibits high thermal stability and specificity for DNA templates.
Automatically generated - may contain errors
Lab products found in correlation
Proteinase k, by Qiagen (1 mentions)
3730 dna analyser, by Thermo Fisher Scientific (1 mentions)
Picogreen assay, by Thermo Fisher Scientific (1 mentions)
Miseq platform, by Illumina (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Chemidoc xrs machine, by Bio-Rad (1 mentions)
Quantitect reverse transcription kit, by Qiagen (1 mentions)
Midori green advance, by Nippon Genetics (1 mentions)
3 protocols using 5prime hotmaster taq dna polymerase
1
MSI Analysis of FFPE Tumor Tissue
2
Analyzing Butyrate-Producing Obligate Anaerobes
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The V4 region (515f.-806r; FWD: GTGCCAGCMGCCGCGGTAA; REV: GGACTACHVGGGTWTCTAAT) of the 16S rDNA gene was amplified using 5 PRIME Hot Master Taq DNA polymerase (Quantabio). Primer construction and amplification followed the Earth Microbiome Project (www.earthmicrobiome.org ) protocol. Amplified barcoded DNA fragments were quantified using a PicoGreen assay (Invitrogen) and equal amounts (ng) of DNA from each sample were pooled. The aggregate pool was sequenced using a V2 2 × 250 kit on the Illumina MiSeq platform (San Diego, CA) at the University of Colorado Cancer Center, Genomics and Microarray Core Facility. 16S rRNA amplicons were demultiplexed using QIIME 1.9.0 and then denoised into ASVs using DADA2. QIIME2 was then used to rarefy to 11,233 sequences per sample, and calculate alpha diversity (Shannon, Faith’s PD, and Inverse Simpson’s) and beta diversity measures (weighted and unweighted UniFrac). ASVs with genus-level taxonomic assignments were filtered against the “List of Prokaryotes according to their Aerotolerant or Obligate Anaerobic Metabolism” and were assigned a binary value for predicted butyrate production and obligate anaerobic growth. Relative abundances for butyrate production and obligate anaerobic growth were calculated by multiplying the binary value by the relative abundance for the microbe within that sample.
3
Quantitative XBP1 Splicing Assay
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Total RNA was isolated from cells by RNeasy® Mini Kit (Qiagen, Hilden, Germany) and 1 µg RNA was reverse transcribed by using QuantiTect® Reverse Transcription Kit (Qiagen). cDNA was amplified by 5PRIME HotMaster® Taq DNA Polymerase (Quantabio, Beverly, MA, USA) using primers specific for human XBP1 resulting in a 200 bp PCR product for XBP1 and 174 bp product for spliced XBP1s (forward primer: 5’-AGAACCAGGAGTTAAGACAG-3’, reverse primer: 5‘-AATACCGCCAGAATCCATG-3’, Eurofins, Luxemburg, Luxemburg). PCR products were separated on a 12% TBE polyacrylamide gel, stained with Midori Green Advance (Nippon Genetics, Düren, Germany), and visualized on a ChemiDoc XRS+ machine with ImageLab software (Bio-Rad). Ratio of XBP1s to XBP1 was quantified by ImageJ. Uncropped gels and respective molecular weight markers are shown in the Supplementary Information.
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