We are trying to detect gene amplification in canine tissue sections (histomorphology is important). The amplified region is about 60 kb in length. What is the best way to prepare the probe, starting from genomic DNA? Should we make it as long as possible (and perhaps fragment it - what would be the best method for this)? Is single stranded DNA probe really much more sensitive than double stranded? Can it be effectively generated by "PCR" using a single primer + labelled nucleotides (perhaps from an amplicon first generated in normal PCR)?
Please read the attached protocol. I hope you can find answers for all your questions.
Thank you, I didn't remember this excellent protocols book. Although it doesn't cover this exact type of experiment, the DNA virus ISH protocol might be applicable.
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