problem in sequencing of gene - (Dec/17/2014 )
i have tried 3 times to send for sequencing of bacteria gene. this gene i have cloned into T-easy vector. and then i sent it to company for checking the sequence is wrong or correct. but even 3 times , i got wrong all of them. in sequence missed 1 or 2 oligonucleotide at different site in gene.
Does anyone know the reason? i need your help.
i would so much appreciate your comment
Are you looking at the called sequence, or at the electropherogram. Mis-called bases are common at the beginning and end of sequence reads, and about 80=120 bp into the sequence read. Usually these can be identified by looking at the electropherogram.
Of course, another possibility is that your organism simply has mutations, which are quite common.
that is sequencing. i did many genes in genome of that organism ( actinomycete) but just only this gene above 1500bp is problem by its sequencing. i tried to change PCR parameters but still got incorrect sequence, and difference colonies are incorrect at the same site of oligonu. maybe i got mutate from PCR product?
I don't understand. If multiple sequencing results for several colonies of your organism all have the same "incorrect" sequence, then it is not the organisms, or the sequencing results that are wrong, but rather your assumptions. Believe the data, not your preconceptions.