The genomic DNA samples from SH-SY5Y and stable transfected TrkA and TrkAIII SH-SY5Y cell lines were extracted using a Promega Wizard Genomic DNA purification kit, as directed (Promega, Madison, WI, USA). The NM_004304.5 (ALK):c.3522C > A (p.Phe1174Leu) mutation [48 (link),49 (link)] was evaluated in an ABI PRISM 310 genetic analyzer (Applied Biosystems, Waltham, MA, USA) via the double-stranded Sanger sequencing of PCR products, which were generated using the Alk-specific primers (5′-TCCTGTTCCTCCCAGTTTAAGA-3′ and 5′-CACTCTTGCTCCTTCCATCC-3′). The PCR products were purified using a EuroSAP PCR Enzymatic Clean-Up Kit as directed (Euroclone, Milan, Italy) and were sequenced using a BigDye Terminator V.2.1. Cycle Sequencing Kit as directed (Thermo-Fisher Scientific, Waltham, MA, USA). The sequencing of genomic ALK DNA in SH-SY5Y, TrkA SH-SY5Y, and TrkAIII SH-SY5Y cells was performed in forward and reverse directions and repeated.
Eurosap pcr enzymatic clean up kit
Manufactured by Euroclone
Sourced in Italy, United Kingdom
The EuroSAP PCR Enzymatic Clean-Up Kit is a product designed for the purification and cleanup of PCR amplification products. It utilizes an enzymatic approach to remove unwanted primers, nucleotides, and other impurities from the PCR reaction mixture, allowing for the recovery of high-quality, purified DNA fragments.
Automatically generated - may contain errors
Lab products found in correlation
Abi prism 310, by Thermo Fisher Scientific (2 mentions)
Wizard genomic dna purification kit, by Promega (1 mentions)
Bigdye terminator v1.1 cycle sequencing kit, by Thermo Fisher Scientific (1 mentions)
Genemapper software, by Thermo Fisher Scientific (1 mentions)
Reliaprep blood gdna miniprep system, by Promega (1 mentions)
Abi prism 3100 genetic analyzer, by Thermo Fisher Scientific (1 mentions)
Gotaq g2 flexi dna polymerase, by Promega (1 mentions)
Jetquick gel extraction spin kit, by Genomed (1 mentions)
3 protocols using eurosap pcr enzymatic clean up kit
1
Evaluating ALK Mutation in Neuroblastoma Cells
2
Genetic analysis of SHOX gene
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Genomic DNA was extracted from whole blood samples using the ReliaPrep Blood gDNA Miniprep System (Promega) following the manufacturer's instructions.
The MLPA analysis for deletions/duplications detection was performed using the commercial kit SALSA MLPA P018‐G1 SHOX probemix (MRC‐Holland) according to manufacturer's instructions. This MLPA test contains 26 probes spaced 0.2–6.7 kb in the coding region and 0.4–338 kb in the non‐coding region. The amplified fragments were analyzed by capillary electrophoresis on an ABI PRISM 3100 Genetic Analyzer with the GeneMapper software (Applied Biosystems). The data analysis was performed by MRC‐Holland Coffalyser v9.4 software.
Sequencing analysis was performed by direct sequencing of the coding exons (1‐6a/6b). Each exon was PCR amplified using GoTaq G2 Flexi DNA Polymerase (Promega; TableS1 ). PCR products were visualized on a 1.5% agarose gel, purified using EuroSap ‐ PCR enzymatic clean‐up kit (Euroclone), and then sequenced in the forward or reverse direction with the BigDye Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems) and analyzed on an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems).
The MLPA analysis for deletions/duplications detection was performed using the commercial kit SALSA MLPA P018‐G1 SHOX probemix (MRC‐Holland) according to manufacturer's instructions. This MLPA test contains 26 probes spaced 0.2–6.7 kb in the coding region and 0.4–338 kb in the non‐coding region. The amplified fragments were analyzed by capillary electrophoresis on an ABI PRISM 3100 Genetic Analyzer with the GeneMapper software (Applied Biosystems). The data analysis was performed by MRC‐Holland Coffalyser v9.4 software.
Sequencing analysis was performed by direct sequencing of the coding exons (1‐6a/6b). Each exon was PCR amplified using GoTaq G2 Flexi DNA Polymerase (Promega; Table
3
Purification and Sequencing of TrkA Exons
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TrkA exon 1–8 and exon 8–17 RT-PCR products were purified from ethidium bromide-stained agarose gels using a Jet Quick gel extraction spin kit, as directed (Genomed, Harrow, UK), cleaned using a EuroSAP PCR enzymatic Clean-Up kit, as directed (Euroclone, Milan, Italy) and PCR amplified using the TrkA exons 1–8 primer set 5′-ATGCTGCGAGGCGGACGGCGC-3′ and 5′-GGAGGCCTGGCCGAAGGGGTT-3′ or the TrkA exons 8–17 primer set 5′-AACCCCTTCGGCCAGGCCTCC-3′ and 5′-CTAGCCCAGGACATCCAGGTA-3′, using a BigDye Terminator V.2.1. Cycle Sequencing kit, as directed (Thermo-Fisher Scientific, CA, USA). Re-amplified products were then sequenced in a mono-capillary DNA sequencer (ABI PRISM 310, Thermo-Fischer Scientific, CA, USA) via double-stranded Sanger sequencing.
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