Taqman pcr master mix reagents

Snap-frozen tissue was proteinase K digested, and genomic DNA (gDNA) was extracted using the Blood & Cell Culture DNA Mini kit (Qiagen, Germantown, MD, USA) as indicated. Isolated gDNA was quantified using the BioTek plate reading spectrophotometer (BioTek Instruments, Winooski, VT, USA). gc distribution in diploid cells were detected and quantified by qPCR using Applied Biosystems 7500 Real-Time PCR Systems with TaqMan PCR master mix reagents (Applied Biosystems, Woburn, MA, USA) and transgene-specific primer/probes.

High titer preparations of AAV9, AAV9-GA, and AAV9-GAST were produced and obtained from the Gene Transfer Vector Core (GTVC) as described above. Five male C57BL/6 mice were each injected with either AAV9, AAV9-GA, or AAV9-GAST containing a self-complementary eGFP transgene under a CMV promoter at a dose of 1.87 × 10¹¹ genome copies per animal by retro orbital administration. After 28 days, animals were sacrificed and tissues were collected and flash frozen. A number of samples were also fixed in 4% PFA before being transferred to a sucrose solution for eventual preparation for histology. To quantify vector abundances in these tissues, the flash frozen samples were homogenized and DNA/RNA were isolated and quantified as described above. To quantify the number of eGFP DNA molecules/cell, DNA isolated from each cell was quantified by ddPCR (QX200 system, BioRad). To quantify the ratio of eGFP RNA molecules/GAPDH, RT-qPCR was performed using an Applied Biosystems 7500 Real-Time PCR System using TaqMan PCR master mix reagents (Applied Biosystems) and transgene-specific primer/probes as previously described.⁸¹ (link)