Editing Generic Transfection Protocol

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.<br><br> 

#The day before transfection, trypsinize and count the cells. Plate 0.5 -1.25x10<sup>5</sup> 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.<br>
#(Optional) The day of transfection, remove growth medium from cells and replace with 0.5 ml of complete growth medium.<br> 
#For each well of cells to be transfected, dilute 0.5 μg of DNA in 25 μl of DMEM<br>
#For each well of cells, dilute&nbsp; 1.5 μl of GeneExpresso Max Transfection Reagent in 25 μl of DMEM.<br>
#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.<br><br>

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

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

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Table 2. Recommended Amounts for Different Culture Vessel.<br> 

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

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[[Category:Protocols]]

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