The biotinylated PCR products of the human PRNP gene were amplified by nonbiotinylated forward and biotinylated reverse primers. The volume of the total PCR mixture was 80 µL, including 2 µL Streptoavidin Sepharose High Performance Medium (GE Healthcare, Chicago, IL, USA), 40 µL PyroMark Binding Buffer (QIAGEN, USA), 8.5 µL high purity autoclaved water and 10 µL PCR products, and shaken for 15 min at 14,000 rpm. The PCR products were washed sequentially with 70% ethanol for 10 sec, PyroMark Denaturation Buffer (QIAGEN, USA) for 10 sec and PyroMark Wash Buffer (QIAGEN, USA) for 10 sec and then mixed with 24.2 µL Pyromark annealing Buffer (QIAGEN, USA) and 0.8 µL sequencing primer (2 µM). Finally, the samples were incubated in a PyroMark Q25 plate holder preheated to 80 °C for 2 min. Prepared samples were loaded into PyroMark Q24 (QIAGEN, USA) and operated in allele quantification (AQ) mode according to the manufacturer’s protocol.
Streptoavidin sepharose high performance medium
Manufactured by GE Healthcare
Sourced in United States
Streptavidin Sepharose High Performance Medium is a product designed for affinity chromatography. It consists of streptavidin, a protein with a high affinity for biotin, immobilized on a Sepharose matrix. This medium is suitable for the purification of biotinylated molecules such as proteins, peptides, and nucleic acids.
Automatically generated - may contain errors
2 protocols using streptoavidin sepharose high performance medium
1
Pyrosequencing of PRNP Gene
2
Pyrosequencing of PRNP Gene
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The biotinylated PCR products of the human PRNP gene were amplified by nonbiotinylated forward and biotinylated reverse primers. The volume of the total PCR mixture was 80 µL, including 2 µL Streptoavidin Sepharose High Performance Medium (GE Healthcare, Chicago, IL, USA), 40 µL PyroMark Binding Buffer (QIAGEN, USA), 8.5 µL high purity autoclaved water and 10 µL PCR products, and shaken for 15 min at 14,000 rpm. The PCR products were washed sequentially with 70% ethanol for 10 sec, PyroMark Denaturation Buffer (QIAGEN, USA) for 10 sec and PyroMark Wash Buffer (QIAGEN, USA) for 10 sec and then mixed with 24.2 µL Pyromark annealing Buffer (QIAGEN, USA) and 0.8 µL sequencing primer (2 µM). Finally, the samples were incubated in a PyroMark Q25 plate holder preheated to 80 °C for 2 min. Prepared samples were loaded into PyroMark Q24 (QIAGEN, USA) and operated in allele quantification (AQ) mode according to the manufacturer’s protocol.
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