Hi,
I have been recently trying to transfect HEK293T with a large plasmid (http://www.addgene.org/53191/) which is around 15 Kb. I am using the PEI method to transfect this DNA. My protocol so far is to mix: 500ug of my DNA of interest, 250 ug of both the psPAX2 and pMD2G into 100ul of Opti-mem. Then a mixture of 100 ul Opti-mem + 15 ul of PEI (1ug/uL)is added to the diluted plasmid mixture. This is then added to a 6-well of HEK293T at 80% confluency. This method has worked for small plasmids but I get very low transfection rates with this large plasmid. Does anybody have experience with transfecting large plasmids?
Any help is appreciated!
Deepak
Larger plasmids are generally harder to transfect. I would change the method. PEI is cost-effective but you may want to try electroporation or a commercial lipid-based kit (e.g. Effectene). Electroporation is usually the most effective.
I've worked with pNL43 which is about 15kb but in HeLa cells primarily. I've had good success with lipofectamine 2000 and JetPrime, but 500ug seems like an excessive amount of plasmid! I typically used 3 ug per well for 6-well plates, collect/ fix 24 hrs post-transfection. Maybe you meant nanograms? If so you can try increasing the amount of plasmid to 1 or 2 ug per well.
You could try Lipofectamine 2000. I see you use really big amounts of your plasmids. We usually use 1,5 ug psPAX, 1 ug MD2G and 2 ug of plasmid of interest. Also 6-well plate with HEKs at ~80% confluency. Good luck!
Please give it a try using following protocol.
You need PEI at 1mg/mL in water at pH 7.0. You need to use 1ug DNA per 1mL culture. You can use either 3 or 5 to 1 PEI:DNA ratio (3:1 being most common). Dilute PEI in 1/20th volume of cell culture in serum free medium. Dilute DNA in 1/20th volume of cell culture in serum medium. Mix the tubes and add diluted PEI to diluted DNA. Mix well and incubate for 30 min. Add the PEI-DNA complex to cells.
- Badges
- Science method