As the primer sequence is all that matters to the PCR reaction, I would use the primers.
Although I am a stranger to species-specific PCR (and therefore I am not sure how reliable 18S would be for this task), I imagine you will be trying to establish which alternative species have the closest match. From that, calculate a primer annealing temperature below the Tm of the primers but above the next-best-match. If there are too many alternative matches with moderate odds of amplifying (ignoring matches that are basically impossible in your sample), maybe try again with primer design. On 9 Jul 2011 17:32, "M Aldeeb" <[email protected]> wrote: > Dear All, > I searched for an 18S rRNA gene of an organism on the NCBI website. I found the sequence and designed primers using the primer tool on the same website. Now, I want to check the primer specificity (i.e. can I use this primer as an identification tool to detect a specific species using PCR) and the question is: > do I need to BLAST the primer sequence (both forward and reverse) or to BLAST the sequence of the amplified fragment (PCR product). Many thanks in advance. > > Best regards > > Mohammad > > Email: [email protected] > Personal Website: http://sites.google.com/site/draldeebsite/ > > > --- On Wed, 7/6/11, mnr mnr <[email protected]> wrote: > >> From: mnr mnr <[email protected]> >> Subject: Blunt end cloning >> To: [email protected] >> Date: Wednesday, July 6, 2011, 1:23 PM >> Hi all. >> >> I have tried cloning a potential toxic gene about 1.2 kbp >> into E.coli with >> T7 promoter. After two months, I am still not successful. I >> am now plannig >> to do cloning into pUC18 vector before subcloning into few >> other vectors >> with different promoters. I have limited available >> restriction sites so that >> I don't have to redesign PCR primers. Is it feasible to do >> blunt-end cloning >> with Phusion polymerase into non-dephosphorylated vectors? >> Will I only be >> getting empty vectors after transformations? >> >> Thank you. >> >> Matt >> _______________________________________________ >> Methods mailing list >> [email protected] >> http://www.bio.net/biomail/listinfo/methods >> > > _______________________________________________ > Methods mailing list > [email protected] > http://www.bio.net/biomail/listinfo/methods _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
