User:Ferdinando Pucci/Notebook/TEMs detection improvement/2008/05/09

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Transduction Titer Yesterday I plated 10^5 bBEND in 2 6-wells costars. filter by gravity the supernatant by linking with sticky tape a 50-ml falcon with a 0.22um filter mounted on a syringe prepare a solution of polybrene 2X in medium (10ml) prepare in a 24-wells costar 540ul of medium for each point of a 10-fold dilution of Tie2GFP (all dilutions start from the 1:2 made adding 500ul of sup to 500ul of medium with polybrene 2X already in the well) add 60ul of vector sup to the 10^-1 dilution, and from this so on in the other dilutions add 500ul of medium with polybrene 2X to the cells and then add the corresponding vector dilution change medium the day after Concentration by ultracentrifugation Done with the same three vectors produced in 15-cm plates (with errors in the transfection protocol): at least 32ml of vector sup each tube (fill with PBS) dry the inner surface of the tube holder to avoid difficulties in removing the tube after the centrifugation 20.000 RPM 2h 20° (balance check at 3.000 and 10.000 RPM) while decelerating stop vacuum quickly empty the tubes in NaClO and put them updown on a paper towel resuspend in PBS avoiding bubbles (300ul for Tie2 vectors, 150 for tetO7-GFP vector) aliquot in 50ul and store at -80°