recently i have been trying to amplify a p450 gene but it kept failing. I was able to amplify a small fragment of the gene but never the whole gene so i decided to go for RACE experiment. With RACE i was able to amplify the gene and then i sent it for sequencing. I have obtained the sequence but both 3' and 5' frangments do not align with the computed sequence i have. Could it be that the sequence was originally not correct? What would be the suggestion? Should i just go ahead and decide new primers? anyone experiencing the same thing before?
Thank you very much
This may just be sequencing artifacts. The very early bases in any sequence are usually rubbish and if the sequence is long then the sequencing signal gets weaker and gets muddled up with background signal and is also poor quality. If you attach a sequence .abi file it may be possible to comment more accurately. Did you sequence in both directions or only one direction?
in addition, P450 is obtained from a huge list of genes of a big family (see part of the list in attachment).
could you refine your request with the right gene name and see if it fits with what you were searching for.
all the best
fred
Paul Rutland
Hi and thank you for your help. I had a question. "if the sequence is long then the sequencing signal gets weaker and gets muddled up with background signal and is also poor quality", you said. why does this happen and which sequences are long for sequencing?
Thank you
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