Problems with T-DNA integration via Agrobacterium - (Dec/16/2006 )
Hi.. guys, is there anybody who is familiar with Agrobacterium-mediated transformation? I just want to clarify facts about T-DNA transferring to the host genome during transformation event. Is it possible not the whole T-DNA integrated into the genome? I coudn't trace one of the genes that should be in the plant genome after transformation. The PCR result is negative. I'm so confused and hope anyone can help me to solve this problem.. 
-alhana-
If I'm not mistaken, it should all be in there.
But: do you have a control to make sure about transformation at all? Have you amplified another gene from the T-DNA with a positive result or do you work with a reporter gene? Are yopr PCR-conditions optimised?
-vairus-
QUOTE (vairus @ Dec 17 2006, 03:57 AM)
If I'm not mistaken, it should all be in there.
But: do you have a control to make sure about transformation at all? Have you amplified another gene from the T-DNA with a positive result or do you work with a reporter gene? Are yopr PCR-conditions optimised?
But: do you have a control to make sure about transformation at all? Have you amplified another gene from the T-DNA with a positive result or do you work with a reporter gene? Are yopr PCR-conditions optimised?
I've transformed 4 transgenes, HPT gene, and GUS gene.I managed to amplify the other three genes, and so did HPT gene. For GUS gene, i've done GUS assay and a lot of them were positive, though not all. For yor info, i used immature embryo as the explant. The PCR-conditions has been optimized, because i followed the protocol for the same gene as my senior did before. Not sure exactly what's the problem..
-alhana-