I used 15 genomic DNA samples and a 1064 bp probe to screen for mating types. The enzyme I used to cut the DNA was HindIII which did not cut (according to Sequencher) the probe. Also, I have sequenced more than 6 clones of the probe and they are all identical.
The protocol I used for digoxigenin hybridization is the standard dig protocol in dig hybridization manual (DNA-DNA). The hybridization temp was 42C.
The probe (~40ng/ml) was produced from the isolate "102" which has this "multiple bands" issue. The other 2 lanes where there is one band are of different species but same genus.
The first photo is the film from the blot and the 2nd is the gel photo. In the film there is an non-homogeneous area but it was probably caused during applying cdp-star.
Any comments welcome.
Hi
The fact that the enzyme did not cut the probe may be due to two things. The first is that the experiment was done in bad condition (digestion temperature, exed in CH3). The second cause may be due to the fact that there is no polymorphic cited for this enzyme in this region of the genome (the region may not be polymorphic if it find a gene in this case it is unlikely to find polymorphism). This explain the fact found identical patterns for clones of the same species and different to the other two.
i expect the probe was to a cdna in which case you expect multiple bands as the enzyme cuts in the introns and parts of the probe binds to different exons in different size fragments of the digested genome. There is no reason to expect the probe to cut with your enzyme but it will stick to any homologous region of the target genome and whether it remains stuck depends on the stringency of the washes and hybridisation
I misunderstood your question Stavros. I think that Artur is correct in his analysis and that degradation of the DNA is the answer
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