3 replies to this topic

[Ninad]

In the reverse transcription protocol, first you add template and random hexamer/oligodT and then incubate at 65/70C
Then you add rest of the stuff to do the reverse transcription.

Can anyone please tell me what is the 65/70C incubation for?

[NKatpase]

Ninad, on Mar 30 2009, 03:12 PM, said:

In the reverse transcription protocol, first you add template and random hexamer/oligodT and then incubate at 65/70C
Then you add rest of the stuff to do the reverse transcription.

Can anyone please tell me what is the 65/70C incubation for?

I would assume that this step anneals the primers to the template, but 65-70C is a bit high. It is usually done at 50-60C for 30 minutes. Depending on the reagents/protocol that you are using, this step may not be necessary.

[Sciurus]

I guess that in this step RNA secondary structures unfold, freeing the primer binding sites and enabling primer annealing.

[molgen]

Sciurus is right, the secondary structures unfold freeing the primer binding sites. The RNA has to be cooled on ice very quickly before you add rest of the stuff to do the tubs. The rapid cooling lets the primer anneal before the RNA refolds.