Transfection of 293-T cells in 6-well plate, using PEI - (Apr/14/2008 )
Hello,
I am trying to transfect 293-T cells in a six-well plate. I used the following method.
Seeded the cells the day before transfection.
Diluted 3ug of DNA in 400ul Optimem. Add 15ul of 1ug/ul PEI. Filter through 0.2 micron. Incubate at room temperature for 20 minutes.
Remove medium from cells. Add DNA/PEI mixture. Place in incubator at 37C for four hours.
Remove the mixture from cells and replace with 600ul medium. Place in incubator.
Harvest the supernatant 3 days later.
I used a method for purifying His-tag protein. I determined absorbance at 280nm and got a negative value.
Can anybody make any suggestions for my transection method? Some of the cells did die because when I pipetted out thee transfection mixture it left a small bare patch on the bottom of the plate. But the rest of the cells don't appear to be dead.
Thank you.
I am trying to transfect 293-T cells in a six-well plate. I used the following method.
Seeded the cells the day before transfection.
Diluted 3ug of DNA in 400ul Optimem. Add 15ul of 1ug/ul PEI. Filter through 0.2 micron. Incubate at room temperature for 20 minutes.
Remove medium from cells. Add DNA/PEI mixture. Place in incubator at 37C for four hours.
Remove the mixture from cells and replace with 600ul medium. Place in incubator.
Harvest the supernatant 3 days later.
I used a method for purifying His-tag protein. I determined absorbance at 280nm and got a negative value.
Can anybody make any suggestions for my transection method? Some of the cells did die because when I pipetted out thee transfection mixture it left a small bare patch on the bottom of the plate. But the rest of the cells don't appear to be dead.
Thank you.
what type of PEI was it? I would omit the 0.2 um filtration step. DNA seems to be low, 5 ug/well maybe? Add FBS to 5% final concentration to the complex/cells during transfection helps. after transfection wait 48hr is good enough. Its lways good to use a reporter construct such as EGFP to test it out, or used as control group.
Thanks for that. Yes, I am planning of doing it with a GFP construct to work out the transfection method.
I used branched 25KDa PEI. I will do as you suggested: omit the filtration step and add 5% FBS to the transfection mixture. Is 4 hours a good period of time to leave it before I remove and replace with medium? Is there a formula for calculating the DNA:PEI ratio?
I used branched 25KDa PEI. I will do as you suggested: omit the filtration step and add 5% FBS to the transfection mixture. Is 4 hours a good period of time to leave it before I remove and replace with medium? Is there a formula for calculating the DNA:PEI ratio?
4-6 hr is fine.
Did you mean charge ratios? 1ug DNA (MW~330/base) has 3 nmole negative charges, and 1 ug PEI (MW~43 per repeating unit) has about 23 nmoles of positive charges. use 1:8-12.5 -/+ ratios for transfection. You may want to titrate the pH of PEI stock to rought neutral with 1 N HCl. good luck.
When using PEI, I've left the transfection solution on the plates overnight without encountering too many problems with cytotoxicity, etc.
Also, when transfecting into 6 well plates, the volume that I've made my transfection solution has been 100ul. I was using CHO cells, but perhaps it is different for 293 cells.