how could one avoid amplification of a very high non-specific band from PCR - (Jun/08/2006 )
i am trying to amplify acp1 gene from cotton, i designed three different sets of primer and each time i observed that a very dense non specific band appear which looks like a specific and desired. each time BLAST analysis and so many optimization were carried out, but failed.
now what can be done now to get rid from this.
It's hard to know without a lot more information. In general, raising your annealing temperature favors specific products. A touchdown PCR would be a good idea for this kind of problem. You mentioned you had Blasted some results -- does this mean you cloned some of the products and sequenced them? Or that you sequenced PCR product directly? Were these sequences similar to ones you want, or junk? Does the gene have multiple copies in the genome? Different genes in the diploid copies? How were your primers designed? I'd recommend Primer3.
Thanks for clarification
Primer sets were designed with the help of DNA STAR software.
The PCR amplified products (one specific 205bp and one non-specific ~300bp) were sequenced. Specific one is matched but non-specifc was not. Even it didn't showed any matching with any other sequence after NCBI BLAST search. Whenever i am trying for optimization, specific one band get diminshes and non-specific one being more denser. The difference in their intensity is approx. of 15 times. Touchdown PCR was also tried but didn't succeeded.
adding 1 base to each of your random primers will decrease your bands to 1/4