IMPORTANT : Preparation of DNA for transfection
We have added additional steps for the preparation of DNA for transfection of 293T cells to optimize efficiency.
These steps not only make the DNA cleaner but also make the final procedure easier by eliminating the use of Falcon 50 ml tubes to wash the DNA pellet, which is quite cumbersome. Instead, we use Eppy tubes. See below.
- after adding 11 ml to the 15 ml of the column eluate, spin the 50 ml tube at 4000 rpm for 30 minutes
- discard the supernatant by inverting the tube
- keep the tube inverted and aspirate all the remaining liquid and droplets on the walls without touching the pellet
- add carefully 500 µl of TE on the pellet without touching it
- let the pellet redissolve for 30 min at RT
- gently pipet up and down until the pelletis totally dissolved, then transfer into a clean 1.7 ml eppy
- add to these 500 µl of TE, 20 µl of NaCl 5M, vortex gently than add 500 µl of isopropanol.
- Mix well without vortexing to generate the DNA jellyfish.
- Quick spin, aspirate the sup and add 500 µl of EtOH 75%
- Mix well on vortex to detach the pellet.
- Spin again
- Aspirate carefully the sup (the pellet is loose so better leave a tad of EtOH)
- Let dry the pellet on the bench.
- Add 100 µl to 300 µl (depending on the expected quantity of plasmid, you want to have a concentration > 1 µg/µl to then adjust to 1 by just adding TE) of TE on the pellet without touching it.
- Let resuspend overnight at +4°C.
- The next day, resuspend carefully until fully homogenous, and run OD to adjust to 1 µ/µl.
- Run RE digest to quality control the plasmid