BBS

50mM BES pH 6.95

280mM NaCl

1.5mM Na₂HP0₄

filter sterilised

(there are 5ml aliquots of BBS in the –20°C freezer in tissue culture)

Protocol:

Day 1:

Plate cells at required density

Day 2:

Transfect in late afternoon/evening. Remember to set incubator to 3% CO₂

Replace medium on cells with DMEM + 10% FBS (I didn’t have antibiotics or non-essential amino acids in this medium).

Mix required amount of siRNA with 0.25M CaCl₂.

Add BBS to a separate tube.

Add siRNA/CaCl₂ to BBS whilst gently vortexing BBS.

Incubate at room temperature for 20mins.

Add dropwise to plates whilst swirling.

Transfer plate to 3% CO₂ incubator.

I used a 10cm dish containing 10ml medium and I used 500µl CaCl₂, 500µl BBS and 0.52nmoles siRNA (final concentration=47nM).

Day 3:

In the morning wash cells once with PBS and add fresh full HeLa medium. Return to 5% CO₂ incubator. After 1 hour wash cells again with PBS and add fresh full HeLa medium. Leave cells in the 5% CO₂ incubator until harvesting (may need to split cells again depending on length of incubation time).

I needed to do a 2^nd transfection in order to improve the knock-down of AP-2. On the evening of day 3 I trypsinised and re-seeded the cells.

Day 4

Transfected cells as before.

Day 5

Washed cells as before.

Day 6

Harvested cells in the afternoon.