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Is there optimal value for firefly and Renilla Luciferase transfection?


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#1 altobarn

altobarn

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Posted 02 June 2009 - 06:56 AM

My colleague was telling me to transfect only 0.0002ng of Renilla (as internal control) into each well of a 24-well plates. Wheareas my firefly luciferase plamsmid I will transfect for 5-100ng. Is this OK? My concern is the renilla amount used would be too low...

Another question is how to decide should I use 24-well plate or 96-well plate to transfect? Is that depends on the total cell number I needed?

Any idea would be very much appreciated

-Altobarn-

#2 KevinK

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Posted 08 June 2009 - 02:10 PM

Hello Altobarn,
Typically the Renilla luciferase is being used as a reference control, so it is recommended that as little Renilla luciferase DNA be transfected as is needed to get a significant signal that is not influenced by any other experimental conditions. The exact amount will depend on the promoter driving the Renilla luciferase, the amount of Renilla luciferase DNA use, optimization of the transfection conditions, and the number of cells used.

As for deciding the well format, determine how many cells or how much lysate you will need to accomplish your experimental goals (for instance, will these samples also be needed for a Western blot?). You might also take your throughput into consideration. If you have lots of samples and can get away with transfections in the 96-well plate, you may be able to use simple add and read product.

I hope this helps you in your research. If you ahve any additional quesitons, feel free to post here or contact Promega technical services.

Regards,
Kevin

Promega Corporation

My colleague was telling me to transfect only 0.0002ng of Renilla (as internal control) into each well of a 24-well plates. Wheareas my firefly luciferase plamsmid I will transfect for 5-100ng. Is this OK? My concern is the renilla amount used would be too low...

Another question is how to decide should I use 24-well plate or 96-well plate to transfect? Is that depends on the total cell number I needed?

Any idea would be very much appreciated

-Altobarn-


Promega Corporation
Madison, WI




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