View source for Transfecting C2C12 Cells

=== Transfecting C2C12 Cells===
* siRNA against Cullin 3 
* siRNA with other sequence

===Materials===
* Two tubes (1.7ml) per well (12 tubes total)
* 6 for control
* 6 for treatment 
* Label tubes 
* Control- tube #1, tube #2
* Cullin 3- tube #1, tube #2
* In tube #1: add 190 ul of DMEM in each
* In tube #2: add 196 ul of DMEM in each

===Method===
# Add 10 ul of pre aliquoted 5 mmol siRNA. Centrifuge, flush tube to mix 
# Add 4 ul of transfection reagent (blue cap) into tube #2 Centrifuge, flush tube to mix 
# *you should have 200 ul total of each solution*  
# Incubate at room temperature for 5 minutes 
# In the meantime prepare 15 ml tubes (1 tube per well)  
# Add 1.6 ml of differentiation media without antibiotic (penicillin/streptomycin)  
# 200 ml of DMEM + 4 ml horse serum  
# In the 1.6 ml of media add 400 ul of the transfection mix (two tubes each with 200 ul)  
# This brings the total volume to 2 ml of solution which is needed for each well to grow 
# Place tube #1 (200ul) into corresponding tube #2, flush to mix, change tips between control and treatment  
# Set tubes at room temperature for 20 minutes 
# Place the 400 ul in each matching tube, flush, and cap 
# Add the new media in the wells (change tips in between) and incubate for 24 hours [do one treatment at a time]  
# Label which wells are control and treatment 
# After 24 hours replace media with normal differentiation media 

'''After the 24 hours'''
# Wash each well 3 times with 2 ml of PBS and remove 
# Add 4% PFA (kept in door) 1 ml per well for 10 minutes, then remove 
# Wash 3 times again with PBS, on 3rd wash leave PBS in dish 
# Plates can be used in next step or stored in the -20 fridge wrapped in parafilm