Transfection of L1HYTK1L Cell
From CCGB
Protocol for Transfection of L1HYTK1L cells (usually RL5)
-- by Hao Wang
Optimizing conditions:
You should first grow cells for two passages in Hygromycin at 1mg/ml to kill all the cells that might have lost the HYTK gene.
Cells should die when plated in 10μM GAN, if not you should optimize the GAN concentration until all the cells die. I use 13μM GAN.
5 days before transfections:
Before each transfections you should grow cells for two passages about 4-5 days in 1mg/ml Hygromycin. In addition, to have very fresh cells you should for the last two or three days feed the cells every day (with warm medium) so that they grow very fast.
Transfection:
Soak the electroporation cuvettes in ethanol and dry them under the hood.
Need 3-4 million cells per transfections, and wash them once with sterile PBS.
Resuspend cells in 0.4-0.8 ml of complete medium (DMEM, 10% BCS, 2% PSF and no additional antibiotics).
Add 200 μg of the plasmid you want to exchange (with Lox1 and 1Lox sites), and 50 μg of CRE expression plasmid, mix.
Incubate at room temperature for 5 min.
Electroporated the cells at 450V, 500μF and 500 msec settings.
Invert the electroporation cuvettes to mix the cells.
Incubate for 10 minutes at room temperature.
Transfer cells to 10ml of warm complete medium and incubate at 37°C for 3-4 days (this gives time for TK protein and mRNA and CRE protein to be eliminated from the cells).
Soft Agar Plating for Colony Isolation:
Count the cells and pellet the amount of cells desired. Wash the cells once with sterile PBS.
Resuspend the cells at 1.8×106 cells/ml in a total of 5 ml rich medium (DMEM, 20% BCS, 2% PSF).
Plate 0.8, 0.4 and 0.2 ml in petri-dishes. The number of colonies differ depending on how healthy the cells are and how they react of electroporation.
Prepare 4% Difco Bactoagar solution and autoclave well in advance. When ready to use microwave it, loosen the cap and keep an eye since it tends to boil very quickly. Need to stop and swirl every 20 sec to make sure that the agar dissolves well. When agar is in solution, place bottle in 55°C water bath for at least 10-15 minutes before using.
Transfer 18 ml Rich medium in a 50 ml conical tube, add 26μl of 10mM GAN, and place in 37°C for at least 10 min (This is enough for 3 plates when plating 6 ml).
Aliquot the appropriate volume of the cells into 60mm plates.
Just prior to plating, remove the 4% agar and the 1ml conical tubes from the water bath, add 2 ml of the agar to the conical tube and mix. Work as quickly as possible since the agar tends to solidify very quickly. If doing multiple plating you should return the agar to 55°C to prevent it from solidifying.
After mixing pipette 5.5 ml of agar/medium mixture into each plate and swirl. Place the plates at room temperature for 10 min and put in the refrigerator for 10 min so that the agar solidifies.
Put the plates in the container covered by aluminum wrap with small holes on top to allow CO2 exchange, include a plate containing water into the container to provide the moisture so that plates do not dry up.
Visible colonies should be ready to be picked after 12 days.
