Sybr safe dna gel stain

Genotyping for the identification of the AGT Met235Thr (rs.699) were performed using fluorescence-based TaqMan^® SNP Genotyping Assays (Applied Biosystems, Foster City, CA, USA). Allele specific probes and flanking primer sets were used along with a pre-made PCR master mix containing ampliTaq DNA polymerase Gold (Applied Biosystems, Foster City, CA, USA) in a reaction volume of 20 μl. PCR consisted of a 10 min heat activation step (95°C) followed by 50 cycles of 15 s at 95°C and 1 min at 60°C. Amplification was performed in PCR thermocycler, Real Time ABI 7500 (Applied Biosystems).
ACE insertion (I) or deletion (D) variants were screened by a polymerase chain reaction (PCR) using a sense primer (5′-CTG GAG ACC ACT CCC ATC CTT TCT-3′) and an antisense primer (5′-GAT GTG GCC ATC ACA TTC GTC AGA T-3′). The PCR product resulted in a 490 bp (I) and 190 bp (D) fragment analyzed on a 2% agarose gel stained with SYBR^® Safe DNA gel stain (Invitrogen).
The presence or absence of repeated sequence of 9 nucleotides of the BDKRB2 polymorphisms were screened by a polymerase chain reaction (PCR) using a sense primer (5′-AGT CGC TCC CTG GTA CTG C-3′) and an antisense primer (5′-TCC AGC TCT GGC TTC TGG-3′). The PCR product resulted in a 89 bp (+9) and 80 bp (−9) fragment analyzed on a 4% agarose gel stained with SYBR^® Safe DNA gel stain (Invitrogen).

Total cellular RNA was extracted using the TRIzol reagent (Invitrogen, Waltham, MA, USA) according to the manufacturer’s instructions. Total RNA concentrations and purity were measured using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). A total of 2 μg of RNA was used for cDNA synthesis. Reverse transcription (RT) was conducted using the Advantage RT-PCR kit (Clontech, Palo Alto, CA, USA). Primers for RT-PCR were purchased from Bioneer (Deajeon, Rep, Korea) for the following genes: GAPDH, Neuro D1, MAP2, Tau, MBP, DCX, NF-L, Wnt, and β-catenin. All primer sequences are summarized in Table 2. The products of the PCR reaction were visualized via electrophoresis in 1.5% agarose gels stained with SYBR Safe DNA Gel Stain (Invitrogen, Waltham, MA, USA). Band images were obtained with a ChemiDoc XRS+ gel imaging system (Bio-Rad, Hercules, CA, USA). Quantitative analysis of the RT-PCR band images was conducted using the ImageJ software (National Institutes of Health, Bethesda, MD, USA).

M. ovipneumoniae inoculum was grown from a previously described nasal wash isolate (MSU NW-4) and expanded exactly as described in the original publication [11 (link)]. Briefly, inoculum was grown at 37 °C in Mycoplasma broth under microaerophilic conditions. On the day of inoculation, media was removed (10,000× g, 10 min, 4 °C) and culture was resuspended in sterile FACS buffer (2% fetal bovine serum and 0.1% sodium azide in DPBS) for counting by flow cytometry [67 (link)]. Cells were subsequently stained with SYBR Safe DNA Gel Stain (Invitrogen, Carlsbad, CA, USA) at 4 °C for 30 min and Absolute Counting Beads (Invitrogen, Carlsbad, CA, USA) were added to solution immediately prior to analysis using an Accuri C6 flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).