Calciumphosphatetransfectionof293T-WestLab.doc

Calcium Phosphate Transfection of 293T I. Reagents/solutions 1. transfection media: 293T cells about 50% confluent. If the cells are too confluent they will not transfect well. You do the transfection in the normal cell medium. 2. 2X HeBs The quality and accurate pH of this solution is of the utmost importance for efficient transfection. We use the highest quality chemicals, and keep them separate from our normal dry chemical stocks. These are labeled tissue culture only and never are any lab implements allowed into the containers. final conc for 200 ml supplier NaCl 274 mM 3.2 g Baker # 3624-05; Mallinckrodt KCl 10 mM 142 mg Mallinckrodt # 6858 Na2HPO4.7H2O 1.4 mM 76 mg Mallinckrodt # 7914; 268g/mol dextrose (D-glucose) 15 mM 540 mg Baker # 1916-01; 180g/mol Hepes (free acid) 42 mM 2 g Calbiochem Ultrol # 391338; 238g/mol - add components to 180 ml. water (we use only ddH2O straight from the purifier, and only glassware that is tissue culture only, never having seen soap) - pH with 1 or10N NaOH (we make special NaOH only used for this) - pH very carefully to 7.02 – use most accurate pH meter you have. - bring to 200mL with ddH20 - remove 25 mL and save - pH remainder to 7.04 (with 1N NaOH); remove 50mL and save - pH to 7.06; remove 50mL and save - pH to 7.08; remove 50mL and save - pH to 7.10; this is the last 25 mL - filter sterilize each pH aliquot separately in cell culture hood - aliquot to 1ml aliquots (eppendorf tubes) - store at -20C; thaw individual aliquots as used. I usually do not reuse unless for nonneuronal cells. - note, as for any CaP transfection, greatest efficiency is obtained with a fine, “sandy” ppt. This is critically dependent on the pH of the HeBS, so it is best to: - use an accurate pH meter - standardize the pH meter (with pH 4.0 and 7.0 standards) repeatedly, until standards are read precisely - as indicated above, make and test multiple batches of 2X HeBS, with slightly different pH’s (eg., pH 7.02-7.10); t