Lipofectin Transfection of Neon. Cardiac Myocytes
A.) DNA precipitation:
1.) Take 15 ug of plasmid DNA , add 5 ul -Gal (5 ug/ul) and fill up to 100 ul with TE.
2.) Ppt with 1/10 NaAc 3M pH 5.2 and 2 vol. ETOH 100% and leave O/N at -20 C.
3.) Spin and wash and dry under flow.
4.) Prepare 2x sterile, polystyrene tubes with snap cap per sample, label 1x with number, 1x with L + number .
5.) Add 1.5 ml Opti-Medium (has 50 ul/50 ml -ME) per tube (prewarmed at 37 C) to labeled tubes and 1.4 ml to L + labeled tubes.
6.) Take 100 ul
7.) Add lipofectin-medium to DNAS-medium to total of 3 ml. Fingershake gently.
8.) Take out 3 pltes with cells from incubator at a time.and pipette off medium with 10 ml pipette.
9.) Then add 3 ml Medium/DNA/Lipofectin.
10.) Incubate at 37 C in CO2 incubator for 5 hours.
11.) Then remove medium and add nutrient medium.
12.) Harvest at 60 hours.
B.) Reagents:
1.) Lipofectin 1mg/ml BRL cat.: 82925 A
2.) Tissue Plates Bio Cat 60 mm Dish Coll. Res. cat.: 40401
3.) Opti-MEM Gibco cat.: 320-1985 AJ
4.) 12 x 75 mm polystyrene sterile tubes Falcon cat.: 2003
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