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Transfection of vectors


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#1 Travis

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Posted 07 March 2009 - 09:18 AM

Hi, I am currently doing luciferase assay. I need to transfect B-gal, PGL-3 construct, PCDNA3 and some other vectors to MDA-MB-231 and T47D human breast tumor cell lines. I have been doing it with the MCF-7 cell line last week and it was totally fine. However this week I follow the same steps with the MBA-MD-231 and T47D cell lines but then I cant get the data. There just simply doesnt have any luciferase and B-gal protein in the cells. What are the possible problems in this? Is it because of low transfection efficiency? How can I optimize the experiment so I can get some data? Thanks very much!!

#2 genehunter

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Posted 07 March 2009 - 02:16 PM

I assume you used the same ratio of total DNA to whatever the transfection agent that you are using for both experiments. I dont have any experience with T47D, but MBA-MD-231 is as readily transfectable as MCF-7 on my hands. Be sure the toxicity is not overwhelming and the cell density is not too high. You can optimize it with fixed amount of cmv-luc vector or egfp vector, change the ratio of vector to transfection reagent. Use cells that are 70%-90% confluent at the time of transfection.

#3 Curtis

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Posted 07 March 2009 - 09:08 PM

I also used lucierase vectors with pcDNA3 and transfected into MCF7....

the process was very easy and I did calcium-phosphate transfection (not lipofectamin) ....in the end I did freeze-thaw to break the cells and extract the proteins (not any lysis buffer)...then I read the signal by a luminometer

I have the protocol somewhere, if you need it just let me know!

#4 Travis

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Posted 08 March 2009 - 05:11 AM

I also used lucierase vectors with pcDNA3 and transfected into MCF7....

the process was very easy and I did calcium-phosphate transfection (not lipofectamin) ....in the end I did freeze-thaw to break the cells and extract the proteins (not any lysis buffer)...then I read the signal by a luminometer

I have the protocol somewhere, if you need it just let me know!



Oh thank you! can you please give me the protocol?? I would love to give it a try... so I just need calcium phosphate to do the transfection?

#5 NemomeN

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Posted 08 March 2009 - 06:29 PM

Not all cells are transfectable with the same efficiency (or even at all). If you still have problems, you can try nucleofection (Amaxa)

#6 Curtis

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Posted 09 March 2009 - 01:35 AM

pm your e-mail to me because I can't send message to you, it says you have disabled your message service or something

Edited by Curtis, 09 March 2009 - 01:46 AM.


#7 cotchy

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Posted 09 March 2009 - 01:41 AM

Curtis could i get a copy of that protocol from you to?

Thanks very much.

Cotchy.

#8 Travis

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Posted 09 March 2009 - 03:09 AM

I assume you used the same ratio of total DNA to whatever the transfection agent that you are using for both experiments. I dont have any experience with T47D, but MBA-MD-231 is as readily transfectable as MCF-7 on my hands. Be sure the toxicity is not overwhelming and the cell density is not too high. You can optimize it with fixed amount of cmv-luc vector or egfp vector, change the ratio of vector to transfection reagent. Use cells that are 70%-90% confluent at the time of transfection.



Hi, may I ask what tranfection agent you use for your experiment?? I am currently using Polyfect from Qiagen and it doesnt work for MDA-MB231, but its alright for MCF-7. Thanks

#9 Curtis

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Posted 09 March 2009 - 03:29 AM

Curtis could i get a copy of that protocol from you to?

Thanks very much.

Cotchy.


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