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

From OpenWetWare
Jump to: navigation, search
Owwnotebook icon.png Project name Report.pngMain project page
Resultset previous.pngPrevious entry      



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°