I digested pcr products by MboI enzyme, expected 2 bands one at 296 and another at 72bp. I got respectively in few samples which means they are resistant, the wild type will not show any cut bands. But I got 3 bands in few samples which have two bands also (296bp+72bp). Is this due to what problem? However I am planning to keep again for the problematic samples.Gel picture as follows: 1 well 100bp ladder, 2 nd well is uncut pcr product,3-12 are resistant type, and 13 is wild type. In few wells there are 3 bands which are highly unexpected.
Did you try adding more enzyme and digesting longer?, since incomplete digestion is the simplest explanation. What DNA are you using as a template for the PCR, could the DNA have a mixture of WT and resistant genotypes?
This may be an incomplete restrictions ...like u may incubate your restriction samples for longer time...
If PCR product concentration is the same in all the digestions, incomplete restriction it's not a good explanation because full digestions are observed in some samples.
I don't know exactly what are you digesting but it seems that some of your samples are heterozygous for this polymorphism and that could be the reason (WT band and resistant bands)
I agree with Alberto they could be heterozygous. It also could be incomplete digestion (don't assume that all the samples are the same). I'm not sure that adding more enzyme is the answer because you also add more glycerol which inhibits the digestion. The vol of enzyme should be no more than 0.1xtotal volume of the reaction. You could add BSA (fraction V, Sigma) to the reaction which tends to soak up the inhibitors and enhance digestion. Also digest for longer time. Water purity can also be a problem, use PCR grade water. You can also put the DNA through a cleanup column. I find that works really well.
Thank you Alberto de Marcos Serrano and Gary Laco . Actually I was provided with pcr products and advised to digest with MBoI , expected only two bands. Afetr the gel result only they cleared the project aim, so it came in light that few types are heterozygous resistant as you believe.so it got three bands and the two bands are actually homozygous resistant. The Enzyme mixture really worked fantasticaaly, so no problem in reaction set up.Thank you all for participating.
it looks like you did get complete cleavage in some samples, and incomplete digestion ( 3 bands) in other samples. The 72 bp band is in many of the samples. It is just faint as expected. It looks like to me that all your clones are positive. In lane 3, for example, there is a lower band than the WT, and a much lower faint band representing the other cleavage product. And this faint band is not in the WT, but is present in all the other samples.
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