1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.
2. To minimize degradation of RNA by RNases, wear gloves when handling samples and reagents, and change gloves regularly while working. Treat water and solutions with DEPC (diethyl pyrocarbonate) to inactivate RNases, and use solutions prepared with RNase-free water and equipment.
3. AMV Reverse Transcriptase is used because it is more stable at higher temperatures than other reverse transcriptases. This allows for the reaction to proceed at 55°C, if so desired, to eliminate potentially inhibitory RNA secondary structures.
4. There is no need to purify the cDNA because the RNase H activity of the AMV Reverse Transcriptase degrades the RNA template.
5. Cycle conditions will depend on your primer, the size of the PCR product, etc. Optimize the PCR for your particular application.
6. Southern Blot analysis can also be performed using an internal gene-specific probe.