Mutational analysis of RET gene was carried out by using standard PCR and Sanger sequencing protocols. Briefly, peripheral blood from MTC patients was collected and DNA was automatically extracted by using MagNA Pure 2.0 (Roche Diagnostics®, Switzerland) platform. PCR was performed with a High Fidelity PCR kit (Roche Diagnostics) following protocol according to Jindřichová et al. [10 (link)]: initial denaturation at 95°C for 10 min followed by 40 cycles (of denaturation at 95°C for 30 s, annealing at optimized temperature (60–68°C) for 30 sec, and elongation at 72°C for 1 min) and final elongation at 72°C for 10 min. The PCR products were evaluated with a Qiaxell system (Qiagen Company®, Hilden, Germany) and mutations were assessed through Sanger sequencing in a 3500 Genetic Analyzer (Life Technologies®, California, United States) by using BigDye 3.1 chemistry.
High fidelity pcr kit
Manufactured by Roche
Sourced in Belgium, Germany
The High-fidelity PCR kit is a laboratory equipment used for the amplification of DNA sequences with high accuracy. It utilizes a high-fidelity DNA polymerase enzyme to ensure precise replication of the target DNA.
Automatically generated - may contain errors
4 protocols using high fidelity pcr kit
1
Genetic Profiling of RET Mutations in MTC
2
ARISA for Microbial Community Analysis
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ARISA was performed using the primers ITSF (5′-GTCGTAACAAGGTAGCCGTA-3′) and ITSReub (5′-GCCAAGGCATCCACC-3′), which amplify the 16S-23S rRNA intergenic transcribed spacers, using 1–5 ng/μl of DNA as input, the high-fidelity PCR kit of Roche (FHIFI ROCHE, Vilvoorde, Belgium) and PCR program as described (Cardinale et al., 2004 (link)). PCR-products were separated using a DNA-1000 chip and 2100 Bioanalyser (Agilent Technologies, Diegem, Belgium). The acquired ARISA data profiles were processed using StatFingerprints in R1 with data normalization and background subtraction.
3
Extraction and Sequencing of mtDNA from Blood
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To extract mtDNA from peripheral blood leukocytes, 2–3 ml of heparin-anticoagulated venous blood was extracted from each leukocyte and purified the mtDNA. GATCCTTGCATGTGTAATCT-3′, the primers were synthesized by the Shanghai Bioengineering Research Centre of the Chinese Academy of Sciences and purified by PAGE. The high-fidelity PCR kit and PCR product purification kit were purchased from Roche. The 1528 bp PCR product nucleic acid fragment was used as the sequencing template. The sequences were sequenced automatically on an ABI prism 3700 sequencers using the dideoxytetra-color fluorescent dye labeling method. For statistical processing, sequencing data were analyzed using DNAStar software. Sequences were proofread using the GenBank H. sapiens mitochondrial genome version with base variation rate = the total number of variant bases/the total number of bases measured × 100% [37 –39 (link)].
4
Plant-optimized sNaVCP gene cloning
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A plant-optimized sNaVCP gene (Genbank accession number GQ389627) from pCRblunt-sNaVCP [22 (link)] was introduced into pICH10990 (ICON Genetics, Halle, Germany) to obtain pICHsNaV. The coding sequence in pCRblunt-sNaVCP was end-tailored to create an EcoRI site at the 5′ end using a high-fidelity PCR kit (Roche) with primers sNaCP-eco (5′-GACGAATTCAACAATGAAGATGGCTTCTAATG) and M13RHT (5′-GGAAACAGCTATGACCATG). The resulting PCR product was digested with EcoRI-SacI and the fragment was ligated into pICH10990 digested likewise to yield pICHsNaV (3′ module). The plasmid was sequenced to assure fidelity and mobilized into Agrobacterium tumefaciens GV3101. The two modules containing the integrase (pICH14011) and the 5′ module (pICH15879) that mediates cytosolic accumulation were coinfiltrated along with the 3′ module [24 (link)]. For expression of NVCP and GFP, the 3′ module vectors pICH-sNVCP and pICH-GFP [21 (link)] were used.
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