Reverse Transcription, Heated Lid yes or no? - Is it necessary to use heated lid during cDNA synthesis incubations? (Oct/19/2007 )
I am using a cDNA synthesis kit where the incubations are:
70 oC for 5 min
25 oC for 5 min
25 oC for 10 min and 37 oC for 1 hour
Reaction stop at 70 oC for 10 min.
I make these incubations in a thermal cycler which has the heated lid possibility, should I use it?
As I have understood heated lid is not good when the incubation temperature is rather low, because it affects the actual temperature of the sample by increasing it.
you kind of answered your own question.
I would not have answered it that way. Heating tubes without a heated lid results in condensation of water on the lid, which changes buffer concentrations dramatically. I would certainly use the heated lid.
yes, but a heated lid for 25C and maybe even for 37C may overheat the reaction. for 70C it is okay.
Well, my thermocycler is programmed to get heat lid always, and we don’t change it when doing RetroTranscription and it always run good.
As you are heating at 70C, you could have condensation, so I’ll better use it.
So..I wonder..in conclusion..which is better..using heated lid or non-heated lid? As both have their advantages and disadvantages...which would you suggest? I would like to know also as I sometimes find it that some people used heated lid and some are not.
Can anybody make me clear? Thanks in advance.
I would say heated lid should be used if your PCR machine is equiped with it. You can adjust the lid temperature to just several degrees higher than the block temperature if possible.