Keep RT mix on ice for 4 hrs - RT-PCR (May/04/2007 )

I am trying to optimize RT-PCR. As a first step, i am trying 4 different temps (37, 42, 45 and 50). My lab doesnt have a gradient thermocycler, so i made a all the solutions before hand and doing RT one by one. That means the sample for 50 deg incubation is sitting on ice for about 4 hrs. Is there going to be a problem of inactivation of reverse transcriptase enzyme? I am using superscriptIII RT from invitrogen.

thanks

---

I would say it definitely won't affect the RT reaction.

---

But the storage condition is -20oC. How would there be no effect on the reaction?
I would prefer to prepare it fresh just before the experiment rather than incubate it on ice, as I am in a hot, tropical country and the ice melt quite rapidly.

---

thanks pcrman for the reply.

virus_fan,

The problem is, i need to thaw all the reagents 4 times or keep the reagents on ice for 4 hrs to make fresh mix. I didnt want that coz there is more danger in doing that than making the mix and storing it on ice. I might be wrong.

---