Transfection conditions for MEF2 RNAi

12/5/05

In general we are transfecting 4-6DIV neurons growing on a monolayer of glial cells.

Remove conditioned medium with a pipette (do NOT aspirate) and save in a 15mL tube. Add an equal volume fresh NB-B27 plus 10uM AraC to the conditioned medium.

Follow the standard CaPO4 transfection protocol. For DNA amounts we use as follows:

There are two conditions (MEF2 RNAi or vector control), you do 6 wells for each condition.

/well 24 well plate

MEF2A RNAi (pSuper vector) 30ng

MEF2D RNAi 30ng

OR use pSuper vector 60ng

Both conditions get

Bclxl (to promote survival) 0.44ug

GFP 0.5ug

(that’s 1ug total DNA/well)

So draw a list

Tube # RNAi (60ng) Bclxl (440ng) GFP(500ng) ddH20 CaCl2 HeBSS

Make master mix for 6.5 wells at 12.5uL total ppt/well

HeBSS (write down pH used) is half the volume in one tube, so 40.6uL

DNA/dd/Ca mix is 40.6uL total in other tube

Calculate volumes of DNA needed. Dilute as necessary for accurate pipetting

Ca is 1/10 final volume, so 8.1uL

ddH20 is variable up to 40.6uL total.

Mix DNA/dd/Ca mix and add dropwise to HeBSS

Wait 20’ for ppt for form

Leave on cells for 22’

Wash 2X with DMEM and 1 x 60’ with BME/10% CS

Then add back CM.