Admin: Created page with "= Protocol = ===== Cell Culture
===== #Prepare a 6-well plate by adding 2 ml of Raw Cell media per well. If coverslips are required, add one coverslip/well prior to addi..."

2021-02-01T19:36:18Z

Created page with "= Protocol = ===== Cell Culture ===== #Prepare a 6-well plate by adding 2 ml of Raw Cell media per well. If coverslips are required, add one coverslip/well prior to addi..."

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= Protocol =

===== Cell Culture =====

#Prepare a 6-well plate by adding 2 ml of Raw Cell media per well. If coverslips are required, add one coverslip/well prior to adding the media. 
#Wash the cells pre-cultured in a T25 flask with 5 ml of PBS and trypsinize with 1.5 ml of trypsin for five minutes. After adding 3.5 ml of fresh media, determine the cell concentration using a hemocytometer. 
#Add the cells to the six well plate at the following concentration:  
#*500,000 cells/well for subsequent microscopy analysis of transfected cells grown on the coverslips => ~35-40 cells/field-of-view at 63x, with ~20% transfected 
#*1 million cells/well for subsequent parachuting of cells onto coverslips
#Rock the plate sideways to evenly distribute the cells.
#Culture the cells overnight at 37oC. 

 

===== Transfection =====

#Thaw the DNA to be used for transfection. 
#Set up a reaction for each well in a microcentifuge tube:
#*Add 3.3 µg of DNA to 150 µl of serum-free media. Vortex the tube for 1-2 seconds to mix.
#*Add the Fugene-HD Transfection Reagent at a ratio of 3:1 of Fugene (µl) :DNA (µg). Flick the tube 15-20 times to mix.   Note:'' The Tranfection Reagent is ethanol-based and evaporates easily. Keep the Transfection Reagent in the fridge until its addition and do not leave uncapped. Re-wrap in parafilm after use. 
#Let the reaction sit for 15 min at Rm temp in the tissue culture hood, for the DNA-containing micelles to form. 
#Add the total reaction volume of each microcentrifuge tube to its respective cell well, drop-by-drop. Shake the plate sideways to mix. 
#Incubate the transfecting cells overnight at 37oC.

 

===== Paraformaldehyde Cell Fixation =====

#Aspirate the media. Wash the cells twice with non-sterile PBS, at 2mL/well. ''Note:'' Be careful not to knock the cells off the coverslips during the wash steps.
#Set up a batch reaction. For six wells, combine:
#*1.2 ml 10X PBS 
#*3 ml 18% PFA 
#*10.8 ml ddH2O
#Add 2mL of reaction/well. Let the plate sit at Rm temp for 15 min. ''Note:'' Keep in the dark if transfected with a fluorophore.
#Aspirate. Wash once-twice with PBS and cover with 2 ml PBS/well.

Raw Cell Transfection - Revision history

Admin: Created page with "= Protocol = ===== Cell Culture ===== #Prepare a 6-well plate by adding 2 ml of Raw Cell media per well. If coverslips are required, add one coverslip/well prior to addi..."

Admin: Created page with "= Protocol = ===== Cell Culture
===== #Prepare a 6-well plate by adding 2 ml of Raw Cell media per well. If coverslips are required, add one coverslip/well prior to addi..."