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°


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