Electrotransfection of BHK-21 Cells

•	Dislodge cells with 1X trypisn/EDTA: 5-10 ml/150 cm2 flat. Incubate 37°C about 5 min. Pipet cells up and down to obtain a uniform suspension. •	Mix with an equal volume of growth medium to stop trypsination. •	Spin 1.5-2 k to pellet cells •	Resuspend in 15ml ice-cold PBS. Spin to pellet (2X) •	Resuspend in 5ml cold PBS. (If it is known that 5ml is more volume than will ultimately be needed, resuspend the cells in an appropriate volume.) •	Count cells using a hemocytometer: 450ul PBS 25ul cell resuspension 25ul Evan’s blue (or Trypan blue) __________________________ 500ul Mix •	Apply about 25ul to hemocytometer. Count 4 squares having 16 sub squares/ square. Calculate average for the 4 squares. -	Calculate: Avg. #cells X 104 X dilution factor (20 here) =#cells/ml •	Adjust cell volume with PBS to obtain 1X107 cells/ml •	Mix 400ul cells with 5-10ug RNA in sterile eppendorf tube. Keep on ice. (Need one 2mm gap cuvette/ electorporation) Electroporate 1 cell sample wo/RNA. •	Electroporation: -	Remove cuvette from ice immediately prior to electroporating. Warm by hand and pulse. -	Pulse conditions: 450 V (450V setting) 720 ohms (R10) 100 uF Pulse length should be about 0.7msec. •	Pulse twice in succession. •	Add 15ml growth media and put into 1 T75 flask. •	Harvest virus (supernatant) at 3+ CPE. Should be between 24-48 hrs.