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More qPCR problem

Suddenly, I feel the pain of qPCR. I did not use any controls before, which is wrong and let me know that I still know very little about qPCR. Juan and Daniel gave me great help on trouble-shooting. I did another experiment with no-reverse-transcriptase control and water control. I found that both control had amplification, indicating that genomic DNA was not eliminated completely even with DNase treatment, and that there are contaminations in some reagents or air flow. So I did following things to remove the contaminants:

  1. Order new primers;

  2. Use all new reagents and water;

  3. Always use filter tips;

  4. Never open a PCR plate or tubes with amplicons that can be templates of your qPCR in the qPCR preparation area;

  5. Use 10% bleach to clean all the pipettes and working area;

  6. Do it in the hood to avoid contaminants in the air flow.

I tested 5 water replicates for each primer after these treatments, but I still could not make all replicates get nothing. Two or three wells still could get some amplification, although very low (Ct > 36). Still need to figure out why.