I am performing real time qPCR and my negative controls are amplifying. Why would this happen?
1) Aliquot your probe and primers into small aliquots with enough product to run only a few experiments. Not only does this guard against contamination but will also help minimize the number of freeze/thaw cycles which degrade oligonucleotide quality.
2) Use separate work areas for qPCR reagent preparation, DNA/template addition and amplification product handling.
3) Clean qPCR work areas and pipettes (designated for qPCR use only) regularly with a DNA degradative agent and follow up with 70% ethanol.
4) Use only sterile, filtered pipette tips to minimize aerosol contamination of the pipettes.
5) If you continue to have trouble, consider using Uracil-N-Glycosylase (UNG) in your assay set up.