Paul.xu: Created page with "Use this procedure to transfect plasmid DNA into HEK 293 cells in a 24-well format (for other formats, see '''Table 2''' below). All amounts and volumes are given on a per well b..."

2014-02-10T00:41:29Z

Created page with "Use this procedure to transfect plasmid DNA into HEK 293 cells in a 24-well format (for other formats, see '''Table 2''' below). All amounts and volumes are given on a per well b..."

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Use this procedure to transfect plasmid DNA into HEK 293 cells in a 24-well format (for other formats, see '''Table 2''' below). All amounts and volumes are given on a per well basis. 

#The day before transfection, trypsinize and count the cells. Plate 0.5 -1.25x105 cells per well in 0.5 ml of complete growth medium. Cell density should be 50-80% confluent on the day of transfection. See '''Table 1''' for other culture vessels. 
#(Optional) The day of transfection, remove growth medium from cells and replace with 0.5 ml of complete growth medium. 
#For each well of cells to be transfected, dilute 0.5 μg of DNA in 25 μl of DMEM 
#For each well of cells, dilute  1.5 μl of GeneExpresso Max Transfection Reagent in 25 μl of DMEM. 
#Mix above diluted solutions, mix gently and incubate '''20''' minutes at room temperature to form DNA- Transfection Reagent complexes.
#After 20 minute incubation, add 50 μl of the DNA- Transfection Reagent complexes directly to each well containing cells and mix gently by rocking the plate back and forth.
#Complexes do not have to be removed following transfection. Incubate the cells at 37oC in a CO2 incubator for 18-24 hours post-transfection before assaying for transgene expression. 

 

Table 1. Recommended number of cells to seed the day before transfection. 

{| width="661" height="217" cellspacing="1" cellpadding="1" border="1"
|-
| Culture vessel
| Surface area per well (cm2) 
| Number of adherent cells to seed
| Volume of medium per well to seed the cells 
|-
| 96-well 
| 0.3 
| 25,000
| 100 μl 
|-
| 48-well 
| 1 
| 75,000 
| 200 μl 
|-
| 24-well 
| 2 
| 125,000 
| 500 μl 
|-
| 12-well 
| 4 
| 250,000 
| 1 ml 
|-
| 6-well / 35 mm 
| 10 
| 625,000 
| 2 ml 
|-
| 60 mm / flask T25 cm2 
| 25 
| 250,000 - 800,000 
| 4~5 ml 
|-
| 100 mm / flask T75 cm2 
| 75 
| 1 x 106 - 2 x 106 
| 8~10 ml 
|-
| 150 mm / flask T175 cm2 
| 175 
| 2 x 106 - 5 x 106 
| 20~30 ml 
|}

 

Table 2. Recommended Amounts for Different Culture Vessel. 

{| width="944" cellspacing="1" cellpadding="1" border="1"
|-
| Culture Dish/Plate 
| Culture Medium 
| Plasmid DNA 
| Volume dilution medium 
| Transfection Reagent GEM 
|-
| 96 well plate 
| 100 μl 
| 100 ng 
| 2 X 10 μl 
| 0.3 μl 
|-
| 48 well plate 
| 200 μl 
| 200 ng 
| 2 X 20 μl 
| 0.6 μl 
|-
| 24 well plate 
| 500 μl 
| 500 ng 
| 2 X 25 μl 
| 1.5 μl 
|-
| 12 well plate 
| 1 ml 
| 1 μg 
| 2 X 50 μl 
| 3 μl 
|-
| 6 well plate 
| 2 ml 
| 2.5 μg 
| 2 X 150 μl 
| 7.5 μl 
|-
| 60 mm dish 
| 5 ml 
| 6.5 μg 
| 2 X 375 μl 
| 20 μl 
|-
| 10 cm dish or T75 flask 
| '''8''' ml 
| 10 μg 
| 2 X 500 μl 
| 30 μl 
|-
| 15 cm dish or T175 flask 
| 20~ 25 ml 
| 25~30 μg 
| 2 X 1.5 ml 
| 75 μl ~90 μl 
|}

 

[[Category:Protocols]]

Generic Transfection Protocol - Revision history

Paul.xu: Created page with "Use this procedure to transfect plasmid DNA into HEK 293 cells in a 24-well format (for other formats, see '''Table 2''' below). All amounts and volumes are given on a per well b..."