substitute for Dnase away - (Feb/15/2008 )
Is there any common lab chemical that one can use to wipe down benches, pippettors, etc., to get rid of Dnases? Don't want to buy Dnase Away if I don't have to.
We don't usually worry much about DNAses. I think that, as long as if you work with gloves and autoclave the stuff you'll be using, it will be fine. For benches and pippetors and such we only clean them with ethanol-soaked paper.
10% bleach is effective but messy. Usually DNAses are not a problem, since DNA is usually stored in TE, which has EDTA in it to inactivate DNAse by chelating the magnesium. And, they can be inactivated by autoclaving.
See my problem is in doing real time PCR. Some of my cDNA samples are turning up negative even though previously (the day before) i got good amplification from them. I aliquot them out when I first make the cDNA, so it's not a freeze-thaw issue. And since the PCR master mix is the same for all the samples, the only thing I can figure is that I'm introducing some DNase somewhere. Hence my question.
How is the cDNA being stored? If you are simply storing the unpurified samples, then I could see this problem happening. At a minimum, add 20 mM EDTA final to the solutions you need to keep around without purification.
The cDNAs are stored at -80 in small (5-10ul) aliquots. I make the cDNAs from purified RNA using MMLV reverse transcriptase and then double the end volume by adding an equal amount of ddH2O water. I haven't been adding EDTA to the samples. The funny thing is that they work perfectly fine in a conventional PCR machine, but as soon as I move to the lightcycler weird things start to happen. But I'll try adding EDTA next time I do the experiment. Thanks for your help.
I've never used a lightcycler, but if I recall correctly, there is a need for slightly more magnesium in the pcr buffers due to the high surface/volume ratio of the capillaries. The EDTA, if you add it, will chelate some of the magnesium in your PCR buffer, so you might want to be aware of that effect and compensate. The amount of chelaation is usually small, since the volume of template added to the mix is small in comparison to the volume of the buffer, but this could become an issue with 20 mM EDTA and 1 ul of template in a 20 ul reaction, which would chelate 1 mM of the magnesium in the buffer, a large and significant effect.
I was thinking about this too, but thanks for reminding me.