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New to sequencing, primer design - (Nov/08/2010 )

I need to sequence a gene of interest that is 681 bp, and it resides in a vector for a total of 3,550 bp. I have a document with the sequence but I want to confirm my sample. I have never designed sequencing primers before and I'm fairly new to the lab, so how would I go about making reliable primers to sequence? I found this site: Should I input my entire sequence? If so what should the distance between primers be? If anyone has any additional advice or help I'd be very thankful, since I have very little idea how to accomplish this goal!


Dear Alkaiser,

this is usually done by using primers that anneal to the plasmid/vector used for cloning. Based on the length you stated I would guess it is a fairy regular cloning plasmid like pUC19 or a derivative thereof. If you bought a cloning kit, there is a good chance that the primers were supplied in the kit. Otherwise look at a vector map for your vector and see what primers are used for sequencing/amplification of your insert. If there really is no primer sequence already published for your vector you can easily upload the entire sequence to BLAST primer and exclude E.coli sequences prior to BLASTing. Then you can choose a primer pair that is fairly close to your the site where you inserted your insert. Seeing as your insert is just shy of 700 bp, you could just about sequence it using only one primer.
Hope it helps,



Thanks! That explanation was just what I needed and I know just how to find primers in the future. Thank you again!