Few days back I isolated some bacteria which i suspected as V.cholerae. But when I extracted their genomic DNA and amplify superoxide dismutase gene by using sodB primer I found something odd. Normally sodB gives a band at 248bp but I got bands at approximately 320. Why do you think this is happening?
How did you isolate the DNA? How did you establish an Annealing temperature for the reaction? Did you use reference strain or clinical isolate?
I did the isolation by boiled DNA extraction method. The annealing temp was set previously. I used V.cholerae N16961 as reference and it gave the band at desired bp.
I examined environmental isolates.
Some questions more :) DNA extraction of the reference isolate is obtained by boiled method too and after PCR the bands from environmental isolates are higher than reference?
DNA isolation of the reference strain was done by phenol-chloroform-isoamile alcohol method, and the band for reference strain was accurate 248bp.
Have you sequenced your inserts to determine if they indeed code for SOD? Your query suggested that you "suspected" your bacteria are V. cholerae. Is it possible that you have either a unique clinical isolate or closely related Vibrio that has a slightly larger sodB gene?
My experience shown that DNA extraction by boiled is not appropriate for some PCR reactions. If the answer of my questions above are YES you have to try the Gradient PCR and if the results are the same try column based DNA extraction methods. If you have the same results you have insertions but 80 bp is too long for that :)
Check The sequence of The product. And blast. That will tell wat u hav got
Sequence is good but it is expensive if you have possibility do it. You must to obtain DNA from samples and reference by one way to make comparison.
The size of the gene you are talking about 248-320bp is not a problem with the boiling method for DNA extraction. Since the primer and PCR conditions worked in your control and not in the test strain . The source of the 248bp may not be Vibrio cholerae since you are working with environmental isolate. Any report of possible inserion in the gene? It will be perfectly okay to sequence the 248bp and check if its sod gene or a truncated sod gene.
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