Exact amplification in negative control???
Posted 21 March 2018 - 10:26 PM
Posted 22 March 2018 - 06:01 AM
It sounds like a mass contamination of possibly multiple sources. This is most likely either from your plasmid positive control, or is from PCR product.
Your best bet is to get rid of all of your PCR stocks, get new everything and start afresh. You should strongly consider (if you can) shifting to a new room, and using a new (only used for mastermix) set of pipettes for setting up the mastermix, then coming back to your old room to add DNA. At a minimum, I would ensure that you have a set of pipettes that you only use for setting up the PCR, and another set that you only use for PCR products.
In my experience, laminar hoods, while useful, do not prevent PCR contamination, and in many cases can actually add to it! This happens because they blow air around - the filters are not capable of removing molecules from the air, so passing more air over your sample increases the chance of having some aerosolized DNA ending up in your reactions.