Guidelines for PCR Optimization using ProtoScript First Strand cDNA Synthesis Kit


RNA Quantification Using Agarose Gel Electrophoresis:
The total RNA concentration and quality can be estimated on a native agarose gel by loading a small sample in 1X RNA loading Buffer next to varying amounts of the included control RNA. The most visible bands are the ribosomal subunits running approximately at 1.8 and 0.9 kb on a 1.2% agarose TBE gel. (Figure 3).

Mix equal volumes of RNA and 2X RNA Loading Buffer, heat for 70°C for 5 minutes, put briefly on ice and run on a 1.5% agarose, 1X TBE gel. Stain and photograph the gel on a UV transilluminator.

2X RNA Loading Buffer:                     
90% deionized formamide
0.05% bromophenol blue

89 mM Tris Base
89 mM Boric Acid
2 mM EDTA (Ph 8.0)