I'd like to design a probe for FISH. The area is non-coding, but the sequence is known. The size of the probe should be 500-1500bp. Do I need to use DOP PCR with the desirable primer? Does anyone has a protocol about it or a guide? Which parameters I need to check on the probe?
A size of 500-1500bp is rather small for doing FISH, the results might not be reliable or the FISH might not work at all with such a small probe.
The probe should not contain repetitive sequences. I would amplify the entire region by PCR and clone it, make plasmid preps, and label the probe by nick-translation incorporating biotin- or digoxigenin-11-dUTP or 16-dUTP using standard protocols.
By the way, which organism are you working on and which material (cell type, tissue, chromosomes, ...) will you use for your FISH?
We are working with human cancer cells. Which is the optimum length? Can I avoid the use of plasmids?I mean that, if it possible to create a long PCR product, which I could label and use it directly as a probe?
You can try the PCR product, but usually, PCR products cause more background and cloning would provide you with an unlimited source. The optimal size of FISH probes depends on the region and what you are looking for and the detection system. 10-20 kb usually work.
I'm thinking of a long PCR and then nick translation of this product. I'll increase the PCR product from 1 to 10Kb.
I'd like to study the LINE transposon, which is also observed in several parts. I'd like to see these several parts.
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