I am attempting to pcr from paraffin sections and have problem to get it to work. I have found a few messages of personal protocols/ideas, etc, in this newsgroup back in the middle of 1990's. The DNA extraction methods are similarly based on removal of paraffin using Xylene/ethanol, Digestion of proteins using proteinase K followed by boiling. I have tried it once and it did not work, probably with insufficient amount of DNA extracted (?), although my positive controls using genomic DNA from other sources were working. I have a few questions:
1. Our samples were formalin fixed, unstained. Does this fixative cross-link DNA and affect the pcr reaction?
2. The proteinase K digestion was carried out in 100 ul, and how much do you use for a pcr reaction in what volume? I've tried 10 uL DNA in a 20 ul reaction and 38 uL in a 50 uL reaction. Should I try less?
3. Does the boiling step completely kill the proteinase K? If not, the DNA prep will contain some active proteinase K that will kill Taq DNA polymerase. If so, I'll probably try 1-2 uL in 50 uL reaction? Can this be a problem?
Comments, suggestions and personal tricks are all welcome.
Dr. Zhonglin Chai
Baker Medical Research Institute, Australia
zhonglin.chai at baker.edu.au