While doing CRISPR cas mediated gene knock out in potato, cloning of vector is done in E coli...so can we directly clone it in Agrobact. and not transforming it to Ecoli.?
Hi Lakshya,
If you are using a binary plasmid to clone your gene of interest into, you can in theory transform this straight into Agrobacterium without going through E. coli first. I would recommend to go through E. coli for many reasons, e.g., in case you want to confirm the integrity of the clone E. coli will give you better yields in plasmid preps and you will also not co-isolate the disarmed Ti plasmid.
Exactly, colony screening is much easier in E.coli. And, secondly agrobacterium transformation needs much higher amount of purified DNA to start with, which would be a mixture instead.
In theory you could go straight to Agro without E coli. But, the efficiency is likely to be low and, as mentioned above, the quality controls steps are easier in E coli than Agro.
While it can be tempting to take shortcuts in protocols, the longer method often gives a better outcome.
Plus, if it doesn't work, what is your next step?
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