11-21-2014, 08:07 PM
Hi everybody,
I'm following an agricultural biotechnology course and I'm wondering if anyone can help me:
We planted floral dip transformed Arabidopsis seeds on a medium containing kanamycin. The transformation (if succeeded) inserted the binairy pXK2FS7 vector, a plasmid containing kanamycin resistance (selection) and a pC aMV 35S - Egfp - gus promotor-gen construct (2 reportergenes). The 35s promotor is flanked by attB1 and attB2 gatewaysites.
If the seeds grow on kanamycin containing medium I come to the conclusion the transformation was succesful. I would expect gfp activity but this was not the case. After two weeks, none of the seedlings were fluorescent under the influence of UV-light. This monday we will check the gus-activity so I don't know about that reportergene yet.
Now I'm wondering why my expectations don't meet reality. Does anybody know why there is no reportergene activity? Are the seedlings to young? Is it the 35S promotor that is not active? If so, why? And how can this be because it is supposed to be strong and constitutive. Or is the promotor active but is there some problem with the mRNA?
Thank a lot for any suggestions,
the biotechnology student
I'm following an agricultural biotechnology course and I'm wondering if anyone can help me:
We planted floral dip transformed Arabidopsis seeds on a medium containing kanamycin. The transformation (if succeeded) inserted the binairy pXK2FS7 vector, a plasmid containing kanamycin resistance (selection) and a pC aMV 35S - Egfp - gus promotor-gen construct (2 reportergenes). The 35s promotor is flanked by attB1 and attB2 gatewaysites.
If the seeds grow on kanamycin containing medium I come to the conclusion the transformation was succesful. I would expect gfp activity but this was not the case. After two weeks, none of the seedlings were fluorescent under the influence of UV-light. This monday we will check the gus-activity so I don't know about that reportergene yet.
Now I'm wondering why my expectations don't meet reality. Does anybody know why there is no reportergene activity? Are the seedlings to young? Is it the 35S promotor that is not active? If so, why? And how can this be because it is supposed to be strong and constitutive. Or is the promotor active but is there some problem with the mRNA?
Thank a lot for any suggestions,
the biotechnology student