During PCR, is there a specific parameter which helps determine the temperature of the heated lid or is this always a fixed value? For example, if I want to do a reverse transcriptase PCR where I only want to incubate at 37C for and hour and heat inactivate at 95C for 5 minutes, do I still need a heated lid or would this only be necessary during a regular PCR? I'm concerned as to whether the heated lid may affect my reverse transcription.
You know when you use a heatblock with no lid and all your sample ends condensing in the lid of the tube? That's why they invented the heated lid. It will discourage your sample to evaporate and condensate on the lid, where the temperature is lower than what you wanted it to be. In the old days of PCR, when heated lids were not available yet, every 95oC cycle would cause evaporation and loss of sample, so after 25 cycles, you had nothing on the bottom of the tube. So they overlaid a droplet of mineral oil on the reaction, so that it wouldn't evaporate, then they had to extract the reaction without the oil, that was probably a big pain! Then a very smart person invented the heated lid, fixing everything.
Use the heated lid, it won't hurt your reaction. It doesn't even need to be a high temperature, just high enough to discourage condensing.
The heated lid exists to ensure that the temperature in the PCR tube is as homogeneous as possible. It should not adversely effect your reaction.
Hey Kunal
for a denaturation temp of 95C for 5 min you will either need to use a heated lid or mineral oil; Otherwise you will lose a lot of your reaction by evaporation
The heated lid will NOT AFFECT your RT: It is simply designed to ensure there is no temp gradient between the top and bottom of your tube and thus minimal loss of reaction by evaporation
By convention you set your heated lid to be +2C higher than your highest temp
Thus for you that would be 97-100C
You know when you use a heatblock with no lid and all your sample ends condensing in the lid of the tube? That's why they invented the heated lid. It will discourage your sample to evaporate and condensate on the lid, where the temperature is lower than what you wanted it to be. In the old days of PCR, when heated lids were not available yet, every 95oC cycle would cause evaporation and loss of sample, so after 25 cycles, you had nothing on the bottom of the tube. So they overlaid a droplet of mineral oil on the reaction, so that it wouldn't evaporate, then they had to extract the reaction without the oil, that was probably a big pain! Then a very smart person invented the heated lid, fixing everything.
Use the heated lid, it won't hurt your reaction. It doesn't even need to be a high temperature, just high enough to discourage condensing.
Heated lid allows you to keep your sample at a homogeneous state and temperature. Without it, heating your sample will create two state: liquid and vapor. However, once you add the heated lid, the heat from the lid will condense the vapor into a liquid state.
nice comments proposed above. my one more question is that while loading PCR reaction for CDNA synthesis, what should be lid temperature, should it be also 2 degree above the maximum temperature to be achieved.
What if you need to run your samples overnight and therefore have a hold temperature of 4 degrees Celsius for after your reaction is complete?
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