Transfection with Linear 25 kDa Reagent (for 6 well dish)

 


1. Seed cells 24 hrs before at 60% confluency (about 2 X 106 cells) since you do
get about some cell death with a 3:1 ratio of the reagent in some cell types.

2. Next day, wash cells 3 – 4 times with cell media shortly before transfecting. 
This is required since there is something that most cells secrete that will interfere
with the reagent.

3. In an eppendorf tube, dilute DNA (3 mg total) in 400 ml of serum free media.

4. Add PEI to the diluted DNA and vortex for 10 seconds immediately.

Optimize for cells by trying 1:1, 2:1, and 3:1 ratios of PEI:DNA; primary cells
will require higher ratios (6:1 to 10:1).

5. Incubate for 15 minutes at room temperature.

6. Add DNA/PEI mix evenly over the 6 well dish containing 2 mls of complete media. 
After adding all the samples, gently swirl plate for a few seconds to mix the components.

7. Next day (give 16h or so), change media and harvest the following day.

8. For most of the cells transfected with my GFP-RASSF1A expression construct,
most are not bright cells, but low expressing cells.

 

Note: For 293 cells and for suspension cells, trypsinize cells as per normal (on the day
of the transfection) and add to 6 well dish in 2 mls of complete media. Carry out the
above protocol and then add to cells in the wells and gently swirl mix.  Allow PEI/DNA
to sit with the cells until the next day and then change the media.

 

DNA used can be from a normal Qiagen prep kit.

 

Cell lines tried and appearance at 48 hours later:


Cell Ratio Death         Trans Efficiency  Cell type/Cancer

COS-7 2.5:1 < 5% > 60%     Kidney

U2OS 2.5:1 10% > 50%     Bone

H1299 2.5:1 10% ~ 50%*     Lung

293 4:1   Good     Fibroblast

CAD 6:1   20% Good     Neuronal

MCF-7 3:1 <10% ~ 40%     Breast

MCF-10A 3:1 <10% ~ 30%     Breast

1o MEF 4:1 – 6:1     0% Low         Fibroblast

NIH3T3 4:1 – 6:1 <10% ~ 15%     Fibroblast

MDCK 5:1 < 5% ~ 50%     Kidney

IM MEFs 3:1 < 5% > 50%*     Fibroblast

HepG2 4:1 None ~ 50%     Epithelial

HeLa 5:1 None > 50%     Epithelial

A549 5:1 < 5% ~ 30%     Lung 


(IM = immortalized)

Earliest detection of GFP positive cells is at 12 hours later 

* Confirmed by FACS

 

 

Reagent:
PEI is polyethyleneimine, a 25 kDa linear from Polysciences. Make up solution at 1 mg/ml in
sterile water, place in 56 oC bath until it goes into solution, neutralize with HCl to pH 7.2 and
filter sterilize using a 0.22 mm filter.  Unused aliquots can be stored at – 80 oC.  For everyday use,
an aliquot can be kept at 4 oC for up to 5months or more so far.

What I have noticed, when you thaw it out from – 80 oC, PEI come out of solution and incubation
at 56oC clears the solution up.  Once clear, you can start using it.  So, remember this for the future.

 

Linear PEI 25 kDa          Polysciences Inc.           2g      23966-2               $45.95