i am starting CDNA synthesis in my lab , i want to know common precautions required for successful results. today i got confused what should be the temperature of lid whether 2 degree above the maximum temperature in protocol / programme or usual temperature 104 degree.
The heated lid doesn't AFFECT your Reaction: 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 temperature. Thus for you that would be 97-100C
a conventional temp of 105 works really fine, keep in mind that you need this temperature to be HIGHER than evaporation point, if not water of reaction will accumulate on top giving you diference of concentration, I strongly disagree with Hussain temperature almost bellow 100 degrees, some of that water will acumulate in the lid, changing concentration of reactants, you should use 105 or above.
a temperature ranging from 102-105 is fine for cDNA syntheis. usually, the 104 degree is required.
- Badges
- Science method