DNAse I - Degrading my DNA (Sep/04/2007 )
I'm using a Kit to extract RNA and it has an "optional" step to add 200ul of (2u/ul) DNase I in the supplied Buffer. However the instructions supplied with the DNase I has separate instructions using EDTA and incubation times upwards to 30mins. Is their a problem with just adding the DNAse I to the filter tube then proceeding to the washing stage or should I incubate it.
Any help would be appreciated.
well you may ask technical service of your kit manufacturer but here some insights.
A dnase works better in solution. I think especially of a buffered pH which may not be applicable on the column.
Second, the DNAse may not access to all the DNA. so it may be a pb.
Finally some manufacturers have upgraded their kits with a DNAse treatment on the silica. (i'm thniking of macherey nagel, but others may have done such modification.
I don't know what your kit mentions for steps after dnase treatment, but maybe there's a phenol chlo step. So if you're thinking your DNA treatment will goes you to loose some RNA, go for a DNAse bound to beads (ambio turbo for example). You treat in 55µl directly and recover 50µl, which are usable directly for qRTPCR, and an agilent check.
I use Qia columns in which DNase treatment is optional. They provide a buffer with which you add the DNase directly on to the membrane and incubate at RT for 15' after which is the normal washing and eluting steps. Works fine for me without loss of RNA.