Fugene 6 transfection efficiency? - (Jan/09/2008 )

I'm transfecting 293T cells with fugene for Co-IP experiments.
But i've been consistantly getting a 30-40% transfection efficiency. any suggestions?

here are my conditions:
Day before plate cells on 10cm dish in 10% serum w/o antibiotics
Next day ~ 50-60% confluent

15ug DNA in 45ul Fugene (1:3 ratio) diluted in serum free media up to a total of 600ul
DNA/fugene incubated at room temp for 20min and transfected drop wise to cells.

I'm wondering if my plasmid size as affecting the efficiency, as my constructs are larger than 8Kb

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I have used larger plasmids for transfection with fugene and they have worked efficiently. I usually found that cell density can affect transfection, but 50-60% seems fine.

How do you prepare DNA? Are they clean preps? Try to precipitate the DNA you have and transfect again. Do you have to use 15ug of DNA?

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The preps should be clean. I did a Midiprep from Qiagen and checked using the Nanodrop. The 260/280 ratio in was in the 1.8-1.9 range.

According to the attached Fugene protocol, it's recomended to use 10ug for a 10cm petridish reaction. this also gave low efficiency, so I increased the DNA to 15ug. This resulted a slightly better efficiency.

Also, if I were to co-transfect 2 plasmids, is it better to transfect 15ug of each plasmid or both plasmids to a total of 15ug, ie., 7.5ug each?
Because when i transfected 7.5ug each, and the efficiency wasn't as good as just 15ug of one plasmid.

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when we transfect 2 or more plasmids, we keep the total DNA same and vary the conc. of individual DNA. like for eg. 2 plasmids, we will use 7.5ug each. Of course, efficiency will be lower but having too much DNA could alter transfection efficiency.

i would suggest you try to optimize transfection in 6 well plate and then once you have it right, you can move to the 10cm plate.

Just to be sure DNA is not an issue here, try to precipitate DNA and transfect it.

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Does the isopropanol and 70% ethanol precipitation step in the midiprep count?

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You may want to do a DNA dosage optimization on these cells in smaller multiwell plates to save reagents. Try to make 100 ul of transfection complexes in opti-MEM with 0.1, 0.2, 0.3, 0.4 ug DNA and 1:3 DNA to reagent ratios, add ~ 20,000 cells in 50 ul of 10% FBS-DMEM per well. you can check if other DNA to fugene ratios by similar way as well.
30-40% rate is very low for this cell line. Also, do you have SV-40 origin/enhancer in your construct, such as pcDNA vectors, that will help in general.

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Precipitate after this step, with 100% ethanol and wash with 70% ethanol. We used to regularly do this extra precipitation step after the isopropanol step with Qiagen kits.

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Yes, the vector backbone is the PCS2+ with a CMV promoter so it should express in 293T.
I've worked with 293T before in my previous lab but with smaller constructs prepared the same way but with the TransIT reagent at similar conditions. Those gave fantastic efficiency.

That's why I'm puzzled as to why I can't get similar efficiencies with Fugene (which is suposed to be the better) using a much larger construct, even when I've factored in size differences by increasing the DNA amt added.

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I agree with scolix, to me DNA size is less a concern. I have done 9kb, to 12 kb plasmid on a regular basis. You statement bothers me: did you use more DNA without compensate it with increased amt of fugene? 293 T is so easy to transfect and you are using one of the best reagent available. Do an optimization, it pays.

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I did increase the amt of fugene when i increased the amt of DNA.

Can I know the DNA to fugene ratios you are using with your 9 to 12kb plasmids? so that I can start from there.

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