Material Supplied
1. First strand cDNA 5ug (lyophilized)
2. beta-actin control PCR mix 200 uL
First strand cDNA is useful for amplifying a particular cDNA using PCR. The PCR reaction must be optimized using varying amounts of the cDNA. This optimization is particularly important when the target mRNA species is of low abundance. The protocol given is for amplifying beta-actin as a control to validate the quality of the first strand cDNA supplied. The PCR conditions to amplify the target cDNA will be based on the primers selected. It should be noted that specific sequence primers as well as degenerate sequence primers can be used successfully to amplify the target sequence.
The first strand cDNA has been prepared from pooled and or amplified mRNA obtained from different tissues. These are not from cultured cell lines. The various tissues vary, but are representative of different organs and tissue types. These include lung, heart, brain, spleen, skeletal muscle, smooth muscle, ovaries, pancreas, liver and kidney. There is lot to lot variation but an overall representation of tissue type is maintained. Oligo dT has been used to prime the synthesis of the first strand using Moloney Murine leukemia Virus (MMLV) Reverse Tranabcedfghase or AMV reverse tranase. The amount supplied is sufficient for at least 50 amplifications. Each lot is tested for amplification of beta-actin cDNA.
