C2C12 TRANSFECTION and STABLE CLONING

  1. Thaw a WCB vials in a T75 flask
  2. Change the medium the day after
  3. Split the cell culture at least a couple of times before plating for the transfection, in order to stabilize it.
  4. The day before the transfection, plate 35000 cell/well in a 6-MW plate with 2 mL/well of complete medium.
  5. The transfection day (avoid to transfect on Friday in order to avoid to work during the week end) prepare 2 x 1.5 mL eppendorf: one for the construct to be transfected and one for the negative control (untransfected). Place 250 uL/tube of Opti-MEM medium and add 2 g of the plasmid DNA only in one eppendorf . Pipet gently to mix completely. Add to both the tubes 7.5 uL of TransIT-LT1 (Mirus) transfection Reagent to the diluted DNA mixture avoiding any contact of the Trans-IT reagent with the sides of the tube. Pipet gently to mix completely and incubate at RT for 30’.
  6. Add the DNA complexes drop-wise to different areas of the wells and gently rock the culture vessel back and forth and side to side to evenly distribute the DNA complexes. Incubate for 24h at 37° C.
  7. After at least 24h split each well in 2 x p100 dishes, diluting 1:15 and 1:25.
  8. Add the right concentration of the antibiotic for the selection of the transfected cells.
  9. From this moment and until the untransfected cells will be all dead, always supplement the culture medium with the right concentration of the antibiotic and split both the transfected and the untransfected control dishes always at the same dilution. Anyway, apply the selection for at least 15 days before plating in the 96 MW for generating stable clones.
  1. To create stable clones, detach the cell culture after 15 days of selection and count the cells.
  2. Take 20x103 cells and bring up to a 4ml final volume with complete medium supplemented with the antibiotic.
  3. Plate 200uL/well in the first column of 2 x 96-MW.
  4. Add 100 uL/well of complete medium supplemented with the antibiotic for the selection of the transfected cells in every other well (columns 2-12).
  5. Transfer with the multichannel 100 uL from the first column to the second one pipetting at least 10 times. Transfer 100 uL from the second column to the third one and go on till the last one. Eliminate 100 uL/well of the cell suspension from the 12th column of each plate.
  6. Incubate ON at 37° C.
  7. The day after, check under the microscope for wells containing a single cell. Those wells have to be checked every day till the cells will reach a 70% confluence. Then split the cells to a 6-MW. When the cells will reach a 70% confluence split them to a T75 flask or to a p100 dish.
  8. Collect RNA, DNA and cryopreserve a couple of vials of each clone in order to test if you obtained any positive clone!