High Pure PCR Template Preparation Kit

Protocol tips

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	Before starting the purification, warm the Elution Buffer to +70°C.

Protocol tips
Before starting the purification, warm the Elution Buffer to +70°C.

Publication protocol

Polymerase Chain Reaction
Genomic DNA was extracted from astrocytes using the High Pure PCR Template preparation kit (Roche Diagnostics, Mannheim, Germany). PCR was performed using specific forward and reverse primers designed for the mitochondrial cytochrome b gene (5′-CAT CAG TCA CCC ACA TCT GC-3′ and 5′-GGT TAG CGG GTG TAT AAT TG-3′) and for the GAPDH gene (5′-CCT TCA TTG ACC TCA ACT ACA T-3′ and 5′-CCA AAG TTG TCA TGG ATG ACC-3′), respectively. Fast Start DNA Master Plus SYBR Green I (Roche Diagnostics) was used with the experimental run protocol: denaturation program was 95 °C for 10 min, followed by 45 cycles of 95 °C for 15", 60 °C for 6′' and 72 °C for 20′'. For evaluation of Bcl-2 expression, mRNA was extracted from astrocytes using High Pure RNA isolation kit (Roche Diagnostics), and cDNA synthesis was performed using the Transcriptor High Fidelity cDNA synthesis kit (Roche Diagnostics). PCR was performed using specific forward and reverse primers designed for the Bcl-2 gene (5′-GGTGGAGGAACTCTTCAGGG-3′ and 5′-GAGACAGCCAGGAGAAATCA-3′) and for the cyclophilin A gene (5′-ATGGCAAATGCTGGACCAAA-3′ and 5′-GCCTTCTTTCACCTTCCCAAA-3′), respectively. Fast Start DNA Master Plus SYBR Green I (Roche Diagnostics) was used with the experimental run protocol: denaturation program was 95 °C for 10 min, followed by 45 cycles of 95 °C for 15 min, 66 °C for 10 min and 72 °C for 15 min.

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Manufacturer protocol

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