Proteinase digestion of tissue for DNA extraction - (Sep/23/2004 )
I want to extract DNA from tumor tissue, and I've tryed with PK but dosent work very well (after on over night incubation at 37 and another 3h at 55 with new amount of protein) I still see the tissue fragments.
I have tryed with tripsine, to disrupt the cells, but that dosent work either.
I dont know what to do any more.... and I need a valid protocol yesterday
PLS help me and Ill be forever grateful
Before you digest your tissue, you have to break up the tissue by mechanical means. First, cut the tissue into small pieces using two scaples or scissors, then break it up further using a homogenizer. In this way, the lysis time will be reduced.
You can also freeze your tissue pieces in liquid nitrogen and then grind to powder before digesting
Why don't you incubate the tissue in the lysis buffer and ProteinaseK at 50 C instead of 37 C. Will do wonders! The optimal temperature for the enzyme is at 50 C and not 37 C. Also, PK is unstable and hence, always better to avoid repeated freeze and thaw. Stock can be 20mg/ml. Use 20-30 ul of it/ tube. If o/n digestion is not complete, spike it with another 20ul or so and leave it for couple more hours. I use waterbath at 50 C. BTW, is it formalin fixed tumor? It's a different story then!
Hope that helps.