I am trying to amplify unknown 5'end of a gene in jute plant by inverse PCR. Everytime I set the PCR and got bands for it. I sent it for sequencing but I found that it is from non target region and only reverse primer is amplifying the bands. What is the solution for that? What criteria should I consider for designing new primers? Should I design nested primers for that? What is the alternative way to reveal the unknown gene sequence?
Nested PCR would definitely be a possible solution for your problem, if you would be using a second inner primer pair that is only amplifying the specific product from the first round of PCR. This is how I typically do inverse PCR myself. Also, I would consider to re-design the forward primer. Maybe it is suboptimal and therefore instead of amplifying the specific product together with your reverse primer, which would "bind" the reverse primer into the correct product, the reverse primer is free to get involved in amplifying unspecific DNA ...
If you know the genome sequence of the plant, use the NCBI design tool to design specific primers. If you don't, you may need to try a lot of different ones, until you randomly find one that's specific enough.
Thank you for responding....what would be strategy to design new primer?...what will be the size of primer?.....
I have worked amplifying 3'-end and 5'-end of a new gene without reference genome and, in brief; I studied restriction sites, designed inverse primers, used some enzymes for restrinction, ligated products and did PCR with the ligation. I obtained more than 1kb in several steps in both, 3' and 5' ends. I could send you slides if you need them.
I hope be helpful
Inverse PCR depends on the size of the product after ligation which in turn depends on the frequency of digestion sites around your gene. From my experience with iPCR it has a limit around 1kb (unfortunately). I normally performed several experiments in parallel using different digestion enzymes. Precipitate your DNA after ligation with addition of co-precipitant (e. g. tRNA) before PCR. Design two pairs of primers (one is nested) be sure that they are as close as possible to the ends of your gene (known sequence). Another powerful approach is gene walking. Check out http://www.ibch.ru/gensensor/Downloads/Chapter2.pdf
Good luck
Many many thanks to you all people for suggestion....I am waiting for your slide Mr. Jaime Jiménez-Ruiz
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