How to decide lid temperature during NGS library preparation when it's not mentioned in the standard protocol.
@Umair
Lid temperature for any PCR/incubation in thermal cycler should be atleast 10°C higher than highest temperature used in the protocol.
Suppose, your protocol has following temperatures;
25° for 15 min
65°C for 15 min
Your lid temperature would be 75°C.
Another example:
95°C for denaturation
56-60°C for annealing
72°C for extension
Your lid temperature would be 105°C.
Dear Umair, we use 105 C lid temp. for amplicon and index PCR in library prep in NGS.
Heated lid option is used for homogenous temperature distribution inside the pcr tube and also to prevent the evaporate the mix. If you are using the kit from the thermo scientific or the Illumina , they always mention to use the heated lid option during library prepration. By default in most of pcr machine it is 105 degree with ramp rate vary with machines.
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