Experiment Gallery
Daily Notes
I did a quick vibes-based agro transform last week. On July 7 I:
- loopful of cells into a tube w/ 50 mM CaCl2
- 100uL per tube (one for +DNA, one control)
- 1.5ul DNA (~500ng) to +DNA tube
- Freeze (as cold as possible, I added CaCl2 to freezer ice + a little 96% ethanol, -10 or -15C, froze pretty fast)
- Thaw 5 mins at 37C
- Add 200uL LB and leave for 20mins to recover
- Plate and spread on plates with Rif. and gent. (maintenence) + kan. (selection for my plasmid)
By the 9th no colonies were visible but the plates had spend some time cold. By the 12th the +DNA plate had 5+ colonies and a big spot of mold. The no-DNA plate had some other contam. This was all a rush job before a camping trip, and checked after we got back - not ideal haha. I streaked 3 colonies onto a fresh R+G+K plate on the 12th and now have plenty growing. I tried an infiltration in tobacco leaves and a yolo duckweed one (just using the dregs in the syringe against some tiny duckweed leaves) - will see if we get transient expression in the leaves, at which point I'll bank my transformed agro and try some proper transformations.
Not a fan of how many steps this is if we want newbies to be able to replicate - lots that needs to go right! Gene gun (order plasmid, shoot, select) would be a lot more accessible. Time to order some algae.
(Inspiration/better protocol: https://pikaard.lab.indiana.edu/protocols/protocols/agrobacterium-growth-and-transformation.html)