sequencing - (Mar/13/2007 )

I have cloned the gene recently and after sequencing i find that there is mutation in the forward primer. I ahve sequenced many colonies and found mutation at the same position . Actually it shows one nucleotide missing. Can anybody face such kind of problem ever.

While I have personally not experienced any seqeunce problems with my primers, there have been occasion and stories on the forum, where the company has made a mistake with the primer it built. The sequence sent to the company was right, but the company itself had either missed or added an extra base.

If this is a real mistake with the primer (and no fault on your own) you can demand the company rebuild your primer. (getting a refund from a company is next to impossible.) Send your sequencing data. Show them the real sequence you ordered. YOu have to be firm because the usual story is that the company will not accept that it has made a mistake. And will try it very best not to rebuild the primer. However if you are firm they will rebuild. (though admiting a mistake is an unlikely outcome.)

We had a similar problem where a single base was missing in one of the primers which we found after sequencing.

So we did the cloning again from step 1 and we sequenced it and it was perfect.