Hi Nadeem, Can you share your primers and the expected sequence from NCBI? It is possible that there are characteristics of either the intended amplicon or the primers you are using that lead to failure of the PCR.
If there is extensive secondary structure in your intended sequence (this can often happen with promoter areas, for example, and some viruses use ribozymes, which will lead to strong DNA secondary structures also in PCR), there are many things you can try to resolve them; solvents, increased annealing temperature, stronger strand-displacement polymerase enzymes, etc.. Best, Cathal On 18/12/12 06:31, Nadeem Ahamad wrote: > Hello, > > I am trying to amplify a gene of interest from a pathogen, but the problem > with the pathogen is that it is a clinical isolate, where the nucleotide > sequence I have taken from NCBI for the same strain /serotype. My primers > are not working on this pathogen. My general doubt is that can the primer > designed for one strain, can also work for other strain of the same species > or same serotype. > > I will be very thankful if anybody can help me in this regard. > > > Nadeem, > > IIT Kanpur, India > > On Tue, Dec 18, 2012 at 12:07 AM, Rachel Schmidt <[email protected]> wrote: > >> I see degradation on my gel that I run to confirm >> purification. >> > > > -- www.indiebiotech.com twitter.com/onetruecathal PGP Public Key: 3B3DE808 (Verify before serious use) _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
