Proteinase digestion of tissue for DNA extraction
Posted 23 September 2004 - 05:38 AM
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
Posted 23 September 2004 - 07:51 AM
Posted 23 September 2004 - 09:12 PM
Posted 27 September 2004 - 02:19 PM
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.