ddNTPs were purchased from Roche Diagnostics (Mannheim, Germany), and exo− Klenow (5000 U/ml) was purchased from Thermo Scientific (Shanghai, China). Inorganic pyrophosphatase (40 U/ml) and Sequenase version 2.0 T7 DNA polymerase (13 U/ml) were obtained from USB Corporation (Cleveland, OH, USA). Pfu PCR MasterMix (2x) was purchased from Solarbio (Beijing, China). Streptavidin-coated Sepharose™ High-Performance beads were purchased from GE Healthcare (Uppsala, Sweden). PyroMark Q24 Advanced Reagents were purchased from Qiagen (Hilden, Germany).
Streptavidin coated sepharose high performance beads
Manufactured by GE Healthcare
Sourced in Sweden
Streptavidin-coated Sepharose high-performance beads are a type of affinity chromatography resin. They are made of cross-linked agarose beads with covalently attached streptavidin, a protein that binds strongly to biotin. These beads are used for the purification and isolation of biotinylated molecules, such as proteins, nucleic acids, or other biomolecules.
Automatically generated - may contain errors
Lab products found in correlation
Pyromark q24, by Biotage (2 mentions)
Pyromark q24 advanced reagents, by Qiagen (1 mentions)
Pyromark gold reagents, by Qiagen (1 mentions)
Vacuum prep workstation, by Biotage (1 mentions)
Pyromark q24 md system, by Qiagen (1 mentions)
Pyromark q24, by Qiagen (1 mentions)
Pyromark wash buffer, by Qiagen (1 mentions)
Pyromark annealing buffer, by Qiagen (1 mentions)
Pyromark denaturation solution, by Qiagen (1 mentions)
Pyromark assay design software, by Qiagen (1 mentions)
Pyromark pcr kit, by Qiagen (1 mentions)
Pyrogold reagents, by Biotage (1 mentions)
6 protocols using streptavidin coated sepharose high performance beads
1
Sanger Sequencing Protocol
2
Pyrosequencing for RAS Hotspot Mutation
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52 specimens were analyzed for hot spot mutations in the RAS genes NRAS, KRAS, HRAS and RRAS-2. PCR amplification primers flank the homologous hotspot regions of these genes. For the pyrosequencing reaction, single-stranded DNA templates were immobilized on streptavidin-coated Sepharose high-performance beads (GE Healthcare, Uppsala, Sweden) using the PSQ Vacuum Prep Tool and Vacuum Prep Worktable (Biotage, Uppsala, Sweden), according to the manufacturer's instructions. Then DNA was incubated at 80°C for two minutes and allowed to anneal to 0.4 mmol/L sequencing primer at room temperature. Pyrosequencing was performed using PyroGold Reagents according to the manufacturer's protocol. Positive and negative controls were used to compare results. Pyrograms were analyzed by PyroMark Q24 software (Biotage), using the allele quantification (AQ) software to determine the percentage of mutant versus wild type alleles according to percentage relative peak height. A sequential nucleotide dispensation protocol was used, reflecting the expected order of nucleotide incorporation and the potential base change within the first, second or third position of the hot spot codons G12, G13 and Q61 of HRAS, NRAS and KRAS. In the same manner codons G23, G24 and Q72 of RRAS-2 were examined. Peak heights are proportional to the number of nucleotides that are incorporated with each dispensation.
3
Pyrosequencing of PCR Products
4
Pyrosequencing for H3F3A and TERTp Mutation Detection
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All samples were analysed using pyrosequencing to detect H3F3A; TERTp mutations were analysed via pyrosequencing for those cases where DNA was available (37/52). PCR primers were designed to amplify the TERTp region containing the C228T and C250T hotspots, corresponding to positions 124 and 146 bp upstream from the ATG site. Primers for the H3F3A mutational analysis were designed to amplify the hotspot codons on exon 2, corresponding to amino acids K27 and G34.
Pyrosequencing analysis was performed immobilising single-stranded DNA templates on streptavidin-coated sepharose high-performance beads (GE Health Care) using the PSQ Vacuum Prep Tool and Vacuum Prep Worktable. Sequencing was done using PyroGold reagents on the PyroMark Q24 instrument (Biotage), according to the manufacturer’s instructions. Negative controls were used to detect background signals. The pyrograms were analysed with the Pyro Mark Q24 software (Biotage) to ascertain the proportion of mutant vs. wild-type alleles according to the percentage relative peak height, as explained elsewhere [18 (link)]. All primers, PCR and sequencing conditions were as described in a previous study [37 (link)].
Pyrosequencing analysis was performed immobilising single-stranded DNA templates on streptavidin-coated sepharose high-performance beads (GE Health Care) using the PSQ Vacuum Prep Tool and Vacuum Prep Worktable. Sequencing was done using PyroGold reagents on the PyroMark Q24 instrument (Biotage), according to the manufacturer’s instructions. Negative controls were used to detect background signals. The pyrograms were analysed with the Pyro Mark Q24 software (Biotage) to ascertain the proportion of mutant vs. wild-type alleles according to the percentage relative peak height, as explained elsewhere [18 (link)]. All primers, PCR and sequencing conditions were as described in a previous study [37 (link)].
5
Bisulfite Pyrosequencing for DNA Methylation Analysis
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Bisulfite pyrosequencing was performed as described previously [26 (link)]. Briefly, bisulfite-treated DNA was amplified using a PyroMark PCR kit (Qiagen, Hilden, Germany). PCR and cycling conditions were according to the kit manual. All pyrosequencing primers (PCR primers and sequencing primers) were based on the selected candidate 450 K array CpG probe using PyroMark Assay Design software (Qiagen). The amplification protocol was performed according to Collela et al. [27 (link)] using a universal primer approach. The biotinylated PCR products were captured using 1.0 μl streptavidin-coated sepharose high-performance beads (GE Healthcare, Little Chalfont, UK). The immobilized products were washed with 70 % alcohol, denatured with PyroMark denaturation solution (Qiagen), and then washed with PyroMark wash buffer (Qiagen). The purified PCR product was then added to 25 μl PyroMark annealing buffer (Qiagen) containing 0.3 μM sequencing primers for specific genes (all primers and their sequences are available on request). Finally, pyrosequencing was performed using the Pyromark Q24 MD system (Qiagen) according to the manufacturer’s instructions using the PyroGold Q24™ Reagent kit (Qiagen). Data were analyzed and quantified with the PyroMark Q24 software version 2.0.6 (Qiagen).
6
Single-Strand DNA Immobilization for Pyrosequencing
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For pyrosequencing analysis, single-stranded DNA-templates were immobilized on streptavidin-coated Sepharose high performance beads (GE Healthcare, Uppsala, Sweden) using the PSQ Vacuum Prep Tool and Vacuum Prep Worktable (Biotage, Uppsala, Sweden), then incubated at 80 C for 2 minutes and allowed to anneal to 0.4 mM sequencing primer at room temperature.
Pyrosequencing was performed using PyroGold Reagents (Biotage) on the Pyromark Q24 instrument (Biotage), according to manufacturer's instructions.
Pyrosequencing was performed using PyroGold Reagents (Biotage) on the Pyromark Q24 instrument (Biotage), according to manufacturer's instructions.
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