rt-PCR false postitives - (Jun/30/2005 )
Hello, I'm doing real-time PCr with SYBR green and I observe a band in the negative control ( with water). I`ve changed everything even the mastermix but nothing works. I've made a gel of the PCR product and they're not primer dimers, sometime they are greater than 900 pb.
Anyone can help me?
If you have some thing (except primer dimer) in your non-template negative control, obviously you have a contaimination.
Have you observe the extra band in you sample well? Is that the same size with your specific band.
If you really want to know where the band derive from, you can cut the band out and send for sequencing.
Hi, i have had a similar problem, the sterile ultra pure water i was using came in glassware that i can only assume through multiple use was contaminate with DNA. To eliminate this problem i have decided to use commerically available PCR grade UPW.
Run your PCR mix (no primers) on a gel, a thermocycled PCR mix (no primers), PCR mix with one primer and PCR mix with another primer, you may find that your primers are the source of contamination depending on the source of the water. If you are using your own sterile UPW you could try exposing it to UV any DNA should be rendered unamplifiable. Also using the tips that are aerosol resistant, work in an airflow and clean surfaces with hypochlorite.