Here I am using recombinant genomic DNA as template. I have transferred the gene via Agrobacterium mediated co-culture method in Tobacco and then I have isolated genomic DNA from transformed leave samples (multiple shoots regenerated on selection media were used). But, even after several trials I am not able to amplify the full length gene (1.7 kb) while internal primer could easily give 240 bp amplicon using the same template. I doubt that the gene has not transferred/integrated completely in the plant genome.
Any views about the integration of truncated gene or any other possible reason behind this ?
Thanks
Several factors may be the reasons for that. For example, the amplification conditions including the temperature and concentration of the added Mg+2 ions and nucleotides. You may also try another type of primers.
Thank you for the suggestions. Actually, the problem is not about the PCR standardization, it is more about the integration of gene in the plant genome. I have amplified the same gene using plasmid DNA as template and it worked fine but here I am using recombinant genomic DNA and its not working. I have tried various polymerases just to make sure that gene size is not the reason. I feel, may be the complete gene has not been incorporated in the plant genome, because I am able to amplify the initial and middle region of the gene, but not the complete gene.
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