I am performing real time qPCR and my negative controls are amplifying. Why would this happen?

The most common reason negative controls come up positive is cross-contamination by a positive control such as a plasmid template. Below are a few suggestions to prevent contamination:

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.