Protocol for siRNA delivery using the targefect siRNA kit
Set
up cells to be transfected so that they are about 70% confluent at
the time of the experiment.
Prepare
transfection complexes as follows:
Use clear plastic tubes for complex formation. If kept frozen thaw the reagents and mix well before use (do not vortex)
Make
aditions as follows: Add Optimem 1 first, then add
dsRNA, mix well by flicking the tube about 12 times to create a
vortexing action. Add
targefect soln A next,
mix well again by flicking the tube and then add targefect
soln B, mix well again.
Incubate the tubes at 37 o C for 25 minutes to form the
transfection complexes.
Wash
cells to be transfected twice with Opitmem 1 . Aspirate the second wash
completely. Add 1 ml of
the transfection mix for 1 well for a 6-well dish (or for a 35 mm
dish). Prepare 250 ul
of transfection complex per well of a 12-well dish, 150 ul per well
of a 24-well dish or 50 ul if using a 96-well plate Incubate the transfection
complexes with the cells at 37 o C for 2 hrs. Add Complete media with
serum (2 ml for a 35 mmm dish or one well of a 6-well plate,
1ml/well for a 12-well dish and 0.5 ml/well for a 24-well dish. Incubate overnight. Replace the media with fresh
complete media the next morning and assy at 24-72 hrs
post-transfection.
When
using fluorescently labed dsRNA we have observed very efficient
delivery into several cell types at 24 hrs post
transfection.
For
testing for silencing of transiently transfected genes we recommend
transfecting the siRNA 12 hrs following transient transfection of
the gene of interest and assaying for silencing of the transiently
transfected gene 24 hrs after transfection of the
siRNA.
For testing silencing of endogenous genes we recommend analysis 48-72 hrs after transfection of the siRNA. | |||||||||||||||