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OUTLINE OF PROCEEDINGS USED IN TISSUE CULTURE TRANSFECTIONS

1. Remove the required cells from liquid nitrogen and thaw at 37°C.

2. Add the thawed cells to 10mls C2GM that has been equilibrated in the 37°C incubator with 10% CO2. Mix evenly and incubate for 24 hours.

3. Split the cells to 1.5 x 105 cells per 100mm culture dish. Use I dish per transfection and one for the negative control. Remember to freeze any leftover cells. Mix evenly and incubate for 24 hours.

4. Transfect the cells (should be ~ 5 x 105 cells per 100mm culture dish). Remember to photograph the cells before transfecting them. Incubate for 24 hours.

5. Photograph the cells and passage 1:20, so that they do not reach confluence during selection. To passage the cells, wash them very thoroughly (about 8 times) in PBS without Ca2+ or Mg2+, add a few drops of trypsin, incubate at 37°C for ~l minute, bash the plate to facilitate cell detachment and add 10mls C2GM. Mix thoroughly and add 0.5mls cell suspension to each of 2() l00mm culture dishes containing 0.5mls preequilibrated C2GM. Mix evenly and incubate for 24 hours at 37°C in 1()% CO2.

6. Feed the cells with selection medium and keep feeding them according to their requirements. (For example, C2 myoblasts require feeding every 2 days).

7. By day 5-6, significant cell death should be seen. From about day 7 onwards, colonies should be visible.

8. When a colony fills the 1()x objective, harvest it and transfer to a single well in a 24well plate containing 1ml selection medium per well. Pick 24 clones altogether, taking no more than 2 clones from any one plate. Only harvest those clones that are well separated from any others.

9. When the clone reaches ~30% confluence, passage into a 60mm culture dish containing 5m1s selection medium.

10. When the clone reaches 30% confluence, passage into 3 x l0()mm culture dishes containing l()mls selection medium per dish. 3 dishes are used so that two can be used for RNA isolation and one for freeze down stocks.

11. When ~30% confluence is reached on all 3 dishes, harvest the cells for RNA and freeze down stocks. Analyse which clones are useful by Northern Blot and transfer these cell stocks to liquid nitrogen. Discard any useless clones (for example, ones which contain the neo insert but not the gene of interest).

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CALCIUM PHOSPHATE TRANSFECTION

All tissue culture operations must be done as sterile as possible in the fume hood. No operations are to be done on the bench. Before any solutions, equipment etc. are taken into the hood, they must be wiped down with 70% ethanol. Benches must be wiped down with Rocall, followed by 70% ethanol, and the hood must be sterilised with UV light for 30 minutes before and after use. Gloves must be cleaned with Hexifoam to ensure sterility and remove residual powder. All solutions must be at 37°C before use.

l. Feed the cells with fresh C2GM before beginning the DNA-Ca solution preparation.

2. For each plate of cells to be transfected, set up two tubes as follows:

Tube l: - l0mg sterile DNA - 63m1 2M CaCl2 - 427m1 TE buffer.

Tube 2: - 500m1 2 x HEPES buffer (in a 15ml Corning tube).

3. Bubble air through the HEPES continuously and while bubbling, add the contents of tube one dropwise to the buffer.

4. Flick the tube a few times to mix the solution. The solution should turn slight[y cloudy, indicating the formation of a fine precipitate.

5. Allow to stand for 3() minutes.

6. Flick the tube a few times to mix the solution and add the precipitate dropwise to the cells. Mix evenly to distribute the precipitate over the entire plate.

7. lncubate for 24 hours at 37°C in 10%C02, after which the cells are ready for splitting, then selection.

Note:

• To sterilise the DNA, ethanol precipitate as normal, but after precipitation, remove the ethanol and wash with 70% ethanol in the fume hood. Air dry the pellet and resuspend in TE buffer to a final concentration of lmg/ml. Usually 50mg DNA is sterilised, with 10mg used in the transfection.

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HARVESTING CLONES USING A CLONING CYLINDER

1. When a single, well-isolated clone fills the field of view from the 10 x objective (but without myotubes being formed in the case of myoblasts), the colony is ready to be harvested. It can actually be seen by the naked eye as a white dot on the underside of the culture dish.

2. For each colony to be harvested, add lml selection medium to a well in a 24-well culture tray and allow to equilibrate in the incubator (37°C, 10%C02).

3. Circle the underside of the plate where the colony is to be picked from.

4. Wash the plates twice with PBS without Ca2+ or Mg2+.

5. Using forceps, place the cloning ring over the clone such that a small amount of vacuum grease allows the ring to stick to the plate, creating a seal.

6. Add 5mls selection medium to the plate to keep the remaining cells alive. Check that no medium has leaked into the clone isolation area.

7. Add a few drops of trypsin to the clone isolation area and incubate for one minute at 37°C.

8. From the appropriate well in the 24-well tray, take some medium and add it to the trypsinised cells. Resuspend the cells thoroughly to ensure that the cells are completely detached.

9. Transfer the cell solution to the appropriate well in the 24-well tray and return it to the incubator.

10. Feed the cells every second day, passaging when they reach 30% confluence.

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FREEZING DOWN CELL STOCKS

This protocol is designed to freeze-down cells used in tissue culture for future use. As with all tissue culture procedures, strerility must always be maintained.

When a clone has reached ~30% confluence in a 100mm culture dish, wash the plate ice in l0mls PBS without Ca2+ or Mg2+ and trypsinise the cells. After incubation at 37°C for approximately 1 minute, bash the plate to ensure all cells are detached. Add 3mls C2GM, mix well and transfer the cell solution to a 1 5ml Corning tube. Centrifuge at 1000rpm for 5 minutes. Remove the supernatant and resuspend the cell pellet in lml freeze-down medium GM + 10%DMSO). Determine the cell count using the haemocytometer so that the number of cells frozen own per vial is known. Split the solution between two cryotubes (0.5ml per vial) and freeze at -80°C. Transfer to liquid nitrogen after 24 hours.

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Notes:

• DMSO is light sensitive and should be kept in the dark.
• The FCS in the C2GM neutralises the trypsin.

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Life Technologies

Product:

Cat. No.: 18324-012 Lot No.: Size: 1 ml, 2 mg/ml Storage Conditions: 4 C

DO NOT FREEZE MIX GENTLY BEFORE USE

hPoFEt~rAMlNE Re~gent is ~ 3:1 (w/w) liposome formultuion of the polyct~tionic lipid 2.3-diokylo~iy-N-12(spermineciubout midokd~yll-N,N-dimethyl.

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