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
