hi, my comment may seem banal but may worth a try. There could be a contamination e.g. with your plasmid. It's atypical to fail with more than one technique I suppose.

Im using Ca phosphate method for transfection get very good result. can you writ the procedure used in ca phosphate method.

Hi Melissa,

At nanoTherics we use magnetic assisted transfection. If you are interested to hear about the technology and how we may be able to help you with transfection, please do not hesitate to contact me by email: [email protected]

Looking forward in hearing from you.

Best Regards,

Aimee-Jayne

Muhammad; this is the protocol we use:

Note: Can change the media 30-60min before transfection to stimulate cell growth and better uptake of the DNA.

1. Dilute stock of 2.5M Calcium Chloride 1:10 in sterile ddH2O.

2. Add 0.5mL to 1.5mL tube. Add appropriate amount of DNA to each.

3. In fresh 1.5mL tubes, put in 0.5mL of 2X HBS.

4. Add CaCl:DNA mix (vortex before adding) into tubes with 2X HBS and vortex briefly.

5. Incubate on ice for ~5min, then mix by pipetting and add drop wise to plates.

6. Mix gently by tilting plate back and forth.

a. Note: I mix the plates several times before changing the media to increase exposure to the DNA precipitate.

7. Change media 4-6 hours after transfection.

Melissa Dobson :

The procedure you used for your transfection almost ok.Just do bit modification i hope you will get good result.

Strtagen ca phosphat kit.

Chang media of Cell line before transfection.The medium should be incomplete(without antibiotic,and FBS)

Dilute desired amount of DNA,(rang 10-30ug) but you used 17ug, dilute in DNA ase free water upto 450ul,if your DNA in TE buffer, Remove theTEbuffer by adding firstly absolute ethanol 1 ml centrifuge discard supernatant than add 70% ethanol again centrifuge, dissolve the pellet with water. Take od.

To 450ul add 50ul cacl2(sol1)pipet it slowly.

Add than 500 ul drop wise vertex it during adding buffer(sol11) Incubate it 10-20 minute at RT)

After incubation add it into T-75 flask, the cells should be 50-60% confluent(avoid over confluent cells keep it 37c, 5% co2.

After 12hr aspirate the medium trpsinz the cells, centrifuge and add the supernatant to stem cells.Keep it for upto 48hr check your flask daily.

Hello Melissa,

Did you get this to work? I am about to start work on 3T3-L1s and am currently looking for good transfection protocols for these hard-to-transfect cells.

Thank you in advance, 

Ralitsa