I have been struggling with PCR for a gene that a former coworker had transformed into Arabidopsis.
About 15 months ago, I was given a bag of Arabidopsis seeds that had been transformed previously by a former lab member. I had isolated what I believed to be a homozygous line for the gene of interest through planting several generations of the plants on herbicide media.
Upon testing for presence of the gene of interest with PCR, I have been unable to get a positive result ever. I have tried adjusting the annealing temp, using different polymerases and kits, and different methods of extracting the genomic DNA. I have used the agrobacterium that was used to transform the plants as a positive control and can almost always get a band.
I understand that the agro has such a high concentration of the GOI and that may be why I am able to get a band but not for the gDNA.
Could someone help me with this issue? Most of the folks in my building are pretty busy with their own projects and don't have time to help me troubleshoot this process. Any insight would be appreciated.