Hello. I just wondering if I have any polymorphic bands on these ISSR-PCR? And reasons for their absence.
'N' are control plants, and I don't see any difference between them and the last samples (which are marked as 'III').
These samples were grown on PGR-containing medium, and there should be at least some polymorphisms. All of these samples are calli on different PGR (I extracted the DNA from the calli. Maybe I shouldn't have done that...).
I tried to analyze the gels with software and they were somewhat identical (excluding N06 and Is). Should I only choose the intense bands and exclude the faint ones? But anyway it makes no sense. Primers are not suitable? But I took them from papers as most effective and sensitive, and PCR conditions have been optimized. Do I really have no polymorphisms after culturing?
There are samples with 'o' and 'I' on the end which are look different. But don't get confused, this was only my 1st attempt to extract plant DNA using CTAB, and it is just didn't come out well.
Vladislav Shakhov For good results with ISSR you should take some other steps:
1) isolate every DNA in 2(3) repeats and make PCR for each one.
2) take DNA for PCR with equal concentration and purity.
3) optimize PCR mix and temperature for most repeatable band profile (Taguchi method is useful)
Generally, ISSR is not the best method for identification mutations caused by PGR - medium. It gave the most conservative patterns on species level among arbitrary-primered PCR methods. You can try ITE-PCR, RAPD, AP-PCR, AFLP for this task. it is useful to use PCR wth 2 different arbitrary primers as well.
Vladislav Shakhov 5 primers for PCR - is not enough to reveal polimorphism of closely related genotypes. You should take first 2-3 samples obviously different and test many primers and primer combinations (you can get 15 for your 5 ISSR primers for instance) for polymorphisms. ITE-PCR can be better because it targets known Transposones.
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