I successfully performed RT-PCR on different cell lines several times, but now it is not working anymore. Some cDNA are amplified while others are not.

So, first I changed the enzymes (either for RT or for PCR) and the respective buffers, I repeated the RT-PCR and I got the same result again: only some cDNA were randomly amplified.

So, that means that the PCR is fine and the problem is only on RNA preparation.

I usually use:

TRIZOL

Chloroform

Isopropanol

75% Ethanol (made with DEPC water)

RT-PCR water to resuspend the pellet

Can the DEPC water used for 75% Ethanol be contaminated with some RNAase?

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