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Calcium phosphate method vs lipofectamine


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7 replies to this topic

#1 EGF

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Posted 28 April 2010 - 05:44 AM

I am having very poor results when transfecting SK-N-SH (neuroblastoma) cells with lipofectamine. I've tried different confluences and DNA:lipofectamine ratios but efficiency is always less than 30-40%.
I don't know if it should be better to try the calcium phosphate method, as I would need a massive amount of plasmid.
Plus, I suspect my transfected protein is involved in calcium pathways and I don't know if it should produce an artifact.
Or perhaps the only solution is to try to make an stable line...
What do you advice me to do?

#2 epibio

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Posted 28 April 2010 - 08:22 AM

Try contacting Mirus Bio regarding your specific cell line. I know that their Trans-It reagents have worked well on other neuroblastoma cell lines.

#3 KevinK

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Posted 28 April 2010 - 09:15 AM

Dear EGF,
There seem to be a few citations that claim to use FuGENE HD for SK-N-SH cells. Promega recently started selling this product and you can obtain a free sample at this address: www.promega.com/transfection. Unfortunatley I'm having a hard time getting any technical details from the papers regarding the DNA:reagent ratio or mass of DNA used, but at least there is an indication of successful transfection.

I hope this can help you in your research. Feel free to respond here or contact Promega Technical Services if we can be of any assitance.

Regards,
Kevin
Promega Corporation
Madison, WI

#4 labrat612

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Posted 28 April 2010 - 12:26 PM

I am currently using Fugene HD to transfect various cell lines (although currently we are not using neuroblastoma cell lines).

For Fugene HD, the product info from Roche does say to test the transfection ratios using approx. 2 ug of DNA.

I'm a little surprised with Lipofectamine not working out well. In my previous life, I routinely used Lipofectamine and Lipofectamine 2000 with a neuroblastoma cell line and with rather descent success. Have you used a positive control to ensure that the lot of Lipofectamine you are using is not a faulty lot?
Check with the Invitrogen technical support to see if they have any ideas.


~Labrat612

#5 EGF

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Posted 28 April 2010 - 10:40 PM

I am currently using Fugene HD to transfect various cell lines (although currently we are not using neuroblastoma cell lines).

For Fugene HD, the product info from Roche does say to test the transfection ratios using approx. 2 ug of DNA.

I'm a little surprised with Lipofectamine not working out well. In my previous life, I routinely used Lipofectamine and Lipofectamine 2000 with a neuroblastoma cell line and with rather descent success. Have you used a positive control to ensure that the lot of Lipofectamine you are using is not a faulty lot?
Check with the Invitrogen technical support to see if they have any ideas.


~Labrat612


In fact Invitrogen says that Lipofectamine 2000 works well with neuroblastoma, and the group that gave me the cells did'nt have problems...With this lipofectamine tube I've also transfected HEK293T and I achieved 90% transfection!!!! (I'm transfecting a GFP fusion protein and is very visual to check the efficiency of transfection).

#6 labrat612

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Posted 29 April 2010 - 06:23 AM

hmmmmm

that is bizarre.

this is a stupid question, bc I'm sure you've already done it: did you follow the same protocol for culturing and transfecting as the group that you received the cells from?

Hmmmmmm...are you using the same plasmid to transfect in HEK293T cells? The fault may lie in the plasmid.

#7 EGF

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Posted 29 April 2010 - 06:38 AM

hmmmmm

that is bizarre.

this is a stupid question, bc I'm sure you've already done it: did you follow the same protocol for culturing and transfecting as the group that you received the cells from?

Hmmmmmm...are you using the same plasmid to transfect in HEK293T cells? The fault may lie in the plasmid.


Yes, I use the same plasmids, and from the same aliquot of midiprep!!

The group that gave me the cells only said me that "transfection worked well with lipofectamine", so I don't ask further details because I was confident and everybody I know using lipofectamine follow the protocol recommended by invitrogen (even reducing to 1/2 or 1/4 the amounts of plasmid and reactive recommended without reducing very much the efficiency!!!).

I've asked for the free sample of Fugene, although in the past I compared the efficiency between Fugene and Lipofectamine in COS, Raw and NIH3T3 cells and Lipofectamine worked better...

#8 labrat612

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Posted 29 April 2010 - 01:10 PM

Well, this is quite the pickle.

I'm stumped. The only thing I can think of is to go back and ask the group what kind of efficiency they were observing. And perhaps, have them go at it with your prep and see if they are doing anything differently.

After that, dude, assume a crap efficiency and head to the bar.

Sorry :P




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