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 freshly obtained Hartley strain guinea pig tissue and appropriately frozen during transportation. Oligo dT has been used to prime the synthesis of the first strand using Moloney Murine leukemia Virus (MMLV) Reverse Transcriptase. The amount supplied is sufficient for at least 50 amplifications. Each lot is tested for amplification of beta-actin cDNA.
1. First strand cDNA 5 ug (lyophilized)
2. beta-actin control PCR mix 200 uL
The ‘First strand cDNA’ is supplied lyophilized. Spin the tube briefly before opening to make sure that the DNA is collected at the bottom of the tube. Reconstitute it in 5uL sterile water.
The beta-actin control PCR mix supplied is ready to use with the supplied first strand cDNA.
Amplification of target sequence cDNA
Amplification of target sequence cDNA requires optimization using varying amounts of the first strand cDNA based on the abundance of the mRNA. Generally 1-5uL of the first strand cDNA is sufficient as the template. It is a good strategy to amplify short segments (200-300 bp) initially, and depending on the amplification results, longer segments could be attempted for amplification. Another proven method is to perform nested PCR using the amplification product of the first PCR.