I have been trying to transform agrobacterium tumefaciens (LBA4404, EHA105 and GV3101) using my plasmid of interest that harbors a kan resistance gene. I got the colonies on plate with antibiotics rif+kan, however when i extracted the plasmid from the colonies and performed restriction digestion using Bsa1 and Sac1, i did not get any band in the samples extracted from colonies, whereas the control plasmid showed 2 bands. I am confused? Any suggestions?
The restriction sites could have been mutated/altered through cloning or maybe this isn't the plasmid you think it is.
Yes, it's true that the transformation has caused loss of restriction site of the RE being used by use for wild type cells. Try with a different RE, it shall work! Best of luck.
Hi Amar,
First you should check your insert(s) in the plasmid and try to do Sanger sequencing or PCR whether your insert(s) is/ are still in the plasmid. Sometime if you cut your plasmid without your insert most probably no band or you got band from vector if it is larger.
- Badges
- Science topic