RT-PCR and PCR

The RT-PCR kit contains polymerase and all necessary reagents to perform single tube RT and PCR. However, we will break the reaction into two discrete steps, RT (reverse transcription to convert mRNA to cDNA) and PCR (to amplify cDNA by the polymerase chain reaction). The RT reaction involves synthesizing cDNA from total RNA or polyA enriched RNA (mRNA). This cDNA is now ready to amplify by PCR. Using specific oligonucleotides (i.e. primers) a fragment of the cDNA can be amplified.

Aim
PCR amplification of the P2X2-specific fragment will be performed using the following primers which introduces restriction sites in order to facilitate manipulation of the amplified DNA product at a later stage (see for example protocol #9).
Forward primer

The original primer pair used for the amplification of P2X2 are described in Brandle, U. et al.(1997). FEBS Letter 404, 294-298, and shown here for reference only.

Reverse transcription (RT)
Materials required
RT-PCR kit (Gibco) Primers (300 nM each)
PCR thermocycler Microfuge
Microfuge 0.5 ml thin walled PCR tubes

Thermocycler Ð Caution Thermocycler can reach temperatures of 95 ℃.

RT set-up
1. To a sterile 500 µl Eppendorf tube add in the following order:

2. Incubate 65 ûC for 5 min.
3. Cool slowly to room temperature .
4. Prepare the following in a separate tube