trouble with PC12 transfection - (Sep/09/2005 )

I have trouble with PC12 transfection with Fugene from Roche.
I've tried transfecting 1 ugr of GFP, which I used in the past with a different cell line successfully, with different ratio and follow every step of the protocol but nothing happened (I got only 2% successes). The DNA is in high quality and the was incubated for 20 min with the Fugene-med. Your comments are much appreciated.

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I'm wondering how your PC12 cells are growing. Usually they grow as big clumbs. Are yours also doing? oOr do you have a trick to grow them up to confluent in your dish?

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I've had success using collagen-coated culture dishes and allowing the PC12 cells to adhere prior to transfection. Forcing them to adhere also largely prevents clump formation, so a greater percentage of your cells will actually be in contact with your transfection reagent. Just make sure to separate them well by thorough triturition prior to seeding the plates.

You might want to consider using a different reagent as well. I've never used Fugene but have used Lipfectamine and Lipofectamine 2000 with good results.

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Elias,
Do you also have experience with retroviral infection of PC12 cells? If yes, what's the infection efficiency?

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Sorry, but I have not tried to infect PC12s.

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Hi Elias,

Where do you buy your collagen-coated plates?

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I make my own with collagen from Sigma - its an insignificant amount of work so the added expense isn't necessary (in my mind). I give a good outline of how this is done here:

http://www.protocol-online.org/forums/inde...indpost&p=23659

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Thanks!

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Our lab uses BioCoat plates from BD (cat # 35 4450) and they work pretty well. Also, we have successfully done transient transfection using CaPO4 method. Good Luck!

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Thanks. I have just ordered them.

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