DeContamination of Buffers ?

[Erdhummel]

Hello everybody !
i try to findout the best way to decontaminate my buffers to get them free of DNA, i tryed 0,2µm filtration with autoclaving - but now i try a following UVtreatment under a LaminarFlow - has anybody any expirience with UVtreatment? Schould i try Sonication to get very small fragments? Is it possible to bye some special Filters? We use Water which is nearly WFI but my NTC are often possitive ;-(
Thanks

[butters]

Hi Erdhummel,

could you please elaborate further. What type of buffers are you refering to? If PCR buffer, why not use the one supply from your Taq? Importantly what PCR detection are you performing?

[Erdhummel]

Hi !
We perform Realtime PCR with TaqMan Probes with special Primers who detects our Gene of Interest and SybrGreen Messurements. I dilute samples with TE-Buffer or ddWater. With the Roche LightCycler480 i use the absplute Quantification (Second Derivative Maximum).
I use 2xIQSybrGreen Mastermix from BioRad and the 2x Platinum QPCR Supermix UDG from Invitrogen. So i have no seperate Buffer supplied by delivering my Mastermixes. The Primers are delivered from Eurofins and they have to be diluted in Water too. I hope you understand my reply ? Thank You..

[Aurora Borealis]

Probably a little too late to answer, but I'll do i anyway.
To avoid contamination we buy our TE-buffer, sterile water and sterile tubes. And if we have to make reagents or dilute our samples, we always use bought stuff free of DNA, DNases and RNases.

But the most important thing is to make sure that you work in a way that protects your samples and reagents from contamination. Work systematically, think about how you use your gloves (should you change them while your working?), think about were and how you treat the samples and reagents (is there a possibilty that you contaminate the reagents while you're working?), is your workstation and pipettes clean?, and so on, and so on... Routines while working are underestimated!

When we have had positive negative controls, it was always because we had contaminated our primers or something like that.