Steven Finkbeiner, Departments of Neurology and Physiology, UCSF

Protocol note: hood scgedule for day of transfection: [2h] - 6h gap - [1h]

CELLS:

Polylysine coat plates.

add 5ml polylysine per 100mm plate.

put at 37℃ overnight(can also do for 3 hours).

remove and save polylysine.

wash plates 2x with 10ml sterile water.

Seed cells:

1/2 X split from confluent plate (approximately 2-5 x 106 cells per 100mm plate) in PC12 culture media.

Incubate overnight in 10% CO2 incubator.

Cells should be 50-70% confluent.

Remove medium from cells and replace with PC12 Transfection Media (10 ml/100mm plate).

Place in 5% CO2 to equilibrate, for approx. 1h (meanwhile, make CaP ppt).

Ca2PO4 PRECIPITATION:

Thaw DNA and 2x HeBS just to room temperature.

Calculate amount of DNA and volumes for ppt.

need 1.4ml ppt/100mm (half is 2x HeBS, half is DNA/CaCl2 mixture).

20-30ug of total DNA required per 100mm plate.

5-20ug reporter plasmid.

3-4ug alpha-globin internal control.

pUC19 to bring to 20-30ug total DNA.

Aliquot volume of 2x HeBS to 1 set of tubes.

Aliquot sterile water to 2nd set of tubes.

To H2O, add the appropriate amount of DNAs.

Add 0.l vol.(of this half) of 2.5M CaCl2 (i.e., 70ul per 100mm plate worth of ppt)。

Using a 5ml pipette, mix DNA/CaCl2.

Transfer this dropwise to 2x HeBS, while gently mixing.

Let sit 20min in the dark; at end, solution should look slightly cloudy.

Add 1.4ml of ppt dropwise to each plate.

Tilt gently to mix.

Put back in 5% CO2.

Incubate 5-7 hours undisturbed.

GLYCEROL SHOCK:

Warm 2xHeBS, PC12 Culture Medium, PBS, 50% glycerol (all sterile).

Make up 25% glycerol/1xHeBS.

will need 2ml per 100mm plate.

1 vol 50% glyc 1 vol 2X HeBS.

typically, room temp when use.

Check plates to confirm normal ppt accumulation.

Aspirate off medium (process 4-5 plates at a time; eg, sets of plates to be compared to eachother).

start timer (set to 3min).

add 2ml glycerol to each plate (start every 15sec);scatter drops,then stack plates and rock to spread.

Let sit 2min.

Under scope cells should appear shrunken.

At 2min aspirate off liquid every 15sec.

Add 4ml PBS (37℃) per 100mm; aspirate, tilting plates to loosen ppt; wash 2x more with 4ml PBS/plate.

Add 10ml fresh PC12 Culture Media; return to 10% CO2 incubator.

Repeat for each set of 4 plates.

Incubate 36-48 hours, analyze.

Reagents:

Polylysine

Sigma

5 mg (one bottle) in 250 ml sterile H2O (tf 20ug/ml) store at 4C, re-use for a few months.

PC12 culture media

DMEM (lab home-made)

10% Horse serum

5% Fetal Bovine Serum (calf serum may also be fine)

2mM glutamine (1/100 from lab stock)

penn/strep (1/200 from lab stock)

PC12 Transfection Media

DMEM (Gibco/BRL # 11960-028; use fairly new bottle so pH is consistent)

10% calf serum

2 mM glutamine (1/100 from lab stock)

penn/strep (1/200 from lab stock)

make fresh, day of transfection

Plasmid DNAs purify by double cesium banding for transient transfections, no need to sterilize the DNA by ethanol pptn

 2X HeBs

 Final Conc  For 2 liters  supplier

 NaCl 274 mM 32 g Baker # 3624-05;

 Mallinckrodt KCl 10 mM 1.42 g Mallinckrodt # 6858

 Na2HPO4.7H2O 1.4 mM 0.76g Mallinckrodt # 7914

 dextrose (D-glucose) 15 mM 5.4 g Baker # 1916-01;

 Mallinckrodt Hepes (free acid) 42 mM 20 g Calbiochem # 910150

 add components to 1.8 l. water

 pH with 10N NaOH, to pH 7.05

 bring to 2l.

 recheck pH; bring to 7.05-7.07

 filter sterilize

 aliquot; parafilm tubes

 store at -20C; thaw individual aliquots as use

 pH is critical; therefore:

 use accurate pH meter

 standardize pH meter (with pH 4.0 and 7.0 standards) repeatedly, until standards are read precisely

 these recipes are for undifferentiated PC12 cells, in 100mm plates

 have also used, per 35mm plate:

 1.5ml Transfection Media

 0.3ml CaP/DNA precipitate

 4 ug plasmid DNA