Greetings...
I am performing experiment of knockdown using siRNA with dual glo luciferase assay system E2920 (promega) and psi-check-TM2 plasmid
1) what instrument should I use for two readings and on what conditions (room temp. etc) to perform the assay?
2) what was the range of the readings of the luminescence (10000 or 20000 or 30000 units) of the both the luciferases and
3)How many times should it be repeated the experiment (if ou have perfomed the same how many times did you repeated the experiment)
4) what volume to use of cells as well as reagents to use for corresponding experiment (e.g 96 well, 12 well, 6 well plates)?
Hi,
You can use plate reader or luminometer to record the luminescent signal.
Other information could be found in the attached file.
Please have a look at it.
On many plate readers you can adjust the sensitivity to obtain the desired range of units for luminescence. I recommend over 1000 units, but less than the capacity of the reader. I performed numerous dual-luciferase experiments with this kit in the past. I used 6-well plates for the transfections, with triplicates of each experiment (3 wells for each condition). For the luciferase read, I used 96-well plates (opaque, of course), and technical triplicates (3 wells each for each well of the 6-well plate, meaning 9 wells per condition).
do you also have difficulties to lyse your cells ?
I use the same kit dual-glo and psiCHECK but to study miRNAs and I have trouble with the lysis step ...
hi Sarah Becker...cells lysis was finely done by the E2920 (promega)-kit..I checked it in microscopic...
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