Transfection of vectors - (Mar/07/2009 )
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!!
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.
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!
Curtis on Mar 7 2009, 10:08 PM said:
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?
Not all cells are transfectable with the same efficiency (or even at all). If you still have problems, you can try nucleofection (Amaxa)
pm your e-mail to me because I can't send message to you, it says you have disabled your message service or something
Curtis could i get a copy of that protocol from you to?
Thanks very much.
Cotchy.
genehunter on Mar 7 2009, 02:16 PM said:
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
cotchy on Mar 9 2009, 02:41 AM said:
Thanks very much.
Cotchy.
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