Hello fellow researchers!
We are trying to transform Arabidopsis with pCAMBIA2022, but we realized that the Chloramphenicol resistance inside the vector will not be compatible with the Agro strain we plan to use (GV3101). I want to insert Spectinomycin resistance (SmR) but I did want to ask about the importance of promoters/terminators and directionality. I found the SmR gene sequence online, and I included an additional ~200 bases upstream which do not appear to be part of the coding sequence. Is anyone familiar with bacterial promoters, and if this sequence contains the correct promoter sequence for expression in both e coli and agrobacterium? Another question is: should a terminator be included as well? Based on what I have read it may not be necessary. Also, does the directionality matter when I clone in the gene. Thank you very much!
To clone Spectinomycin resistance (SmR) into the pCAMBIA 2022 vector, you can follow a general cloning strategy that involves several steps. Here's an overview of the cloning process:
Once you have successfully cloned the SmR gene into the pCAMBIA 2022 vector, you can proceed with your transformation experiments in Arabidopsis using the modified vector.
Please note that this is a general outline of the cloning process, and the specific details and protocols may vary depending on the restriction enzyme sites, primers, and other considerations. It's important to refer to the original research paper or the vector manufacturer's instructions for the specific cloning strategy and protocols recommended for the pCAMBIA 2022 vector.
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