Passive lysis buffer for luciferase assays - (Dec/19/2005 )
Hi All
I am doing dual luciferase assays for the first time
when I put the passive lysis buffer on my cells
i saw within 3-4 minutes white thread like particles
is that normal?
do I need to centrifuge before proceeding to the assay
please answer soon
thanks
Yes you need to centrifuge.
collect the lysate into a eppendorf tube, vortex for 10-15", then centrifuge at 12,000xg for 15" (at RT) or 2 min (at 4C). Transfer the supernatant for assay.
collect the lysate into a eppendorf tube, vortex for 10-15", then centrifuge at 12,000xg for 15" (at RT) or 2 min (at 4C). Transfer the supernatant for assay.
thanks for the answer
I have another question for you
I have done an experiment like the following for co-transfections
pGL3-basic + TK-renilla(pHRG-TK)
A +TK-renilla
B+TK-Renilla
My first question is
Do I need any other controls?
My second question is
A and B are two independent constructs having exactly the same putative region cloned into pGL3
inspite of this ..why do I get ratios that differ 5 fold for the same concentration of Aand B?
thanks
My first question is
Do I need any other controls NO..The renella is the internal control.
I do not understand the second question.
AS for ur first post on the thread like things..it is normal. I usually lyse my cells for 15 mins...
Dear Watson,
with regards to your original question ... the white strands are not unusually and you do not have to clear the supernatant. If you use a 96-well plate system, you have no option to centrifuge and transfer the lysate yet you get the same result as if the experiment was performed in a larger surface area.
with your control question ... if you want transfection controls then the renilla +/- pGL3-basic is fine. If you want luciferase assay controls, then you'll need i) pGL3, ii) renilla and iii) pGL3+renilla to give you i) luciferase alone, ii) renilla alone and iii) both together.
With your plasmid question ... if you are seeing differences in the two constructs then this could be a reflection of how your cells process them more than the actual contructs. Are you sure the constructs are the same? (i.e. have you sequenced them).
Hope this helps,
AussieUSA.
thanks for all the responses
I have some more questions
Please take a look at the folllowing dual luciferase assay readings (I have attached word document)
I have maintained the DNA(ug) : lipofectamine(uL) ratios to 1:2
A and B are two independent constructs ..supposed to be the same sequence
Vectors transfected firefly renilla ratio
TK-Renilla (0.5ug) 76 57636950
TK-Renilla (0.5ug) 80 54768130
basic-pGL3 (2.5ug) + TK-Renilla (0.5ug) 27000 11150773 0.0024
A (2.5ug) + TK-Renilla (0.5ug) 20674 196223 0.1054
B (2.5ug)+ TK-Renilla (0.5ug) 768524 1692291 0.4541
basic pGL3 (5ug) + TK-Renilla (0.5ug) 33124 3161413 0.0105
A (5ug) + TK-Renilla (0.5ug) 50647 259431 0.1952
B (5ug)+ TK-Renilla (0.5ug) 497770 736412 0.6759
Untransfected 207 300 0.69
Why is the renilla value different for each transfection although I have transfected equal DNA concentrations of renilla
Is this too much DNA for 12 well plate?
Does an increase in ratio ( from basic to A or B ) in this case reflect an increase in promoter activity
why does the basic pGL3 show firefly luciferase activity?
thanks
please see the attached word file
thanks
why is your untransfected control showing such a high number?? My untransfected cells control actual numbers are 0.11:0.33 (Lucif: Renella). I think there is a problem of autoflourescence or something.... Your untransfected control should not give you such high numbers
Vectors transfected Firefly Renilla Ratio
Untransfected 207 300 0.69
I dont think it is your DNA conc. I use 0.8 ug/well for 24 well plates with lipofectamine. However, my renella is normally 1/20th of the luciferase concentration. Also I use 2 ul of lipofectamine/well and incubate for 4-6 h. Pull off the media and let it quilibrate for 12-24h.
The basic PGL3 will show luciferase activity. It is often used for experiments involving gene delivery or testing transfection agents. Dont worry about that.
The renella values will vary from well to well due to different number of cells etc. YOu need to do triplicates and only worry about the ratio. As long as you get good numbers you are fine.
Does an increase in ratio ( from basic to A or B ) in this case reflect an increase in promoter activity
No. I dont think so. ..This is becoz the basic PGL3 control vector does not have a promoter. It is used for determing transcriptional activity.
If you have any questions regarding this try contacting Promega's technical team. They make the best PGL3 control vectors.
Thanks for such a detailed response..i a m a beginner and ur advice is great help for me
"Also I use 2 ul of lipofectamine/well and incubate for 4-6 h. Pull off the media and let it quilibrate for 12-24h. "
In the above steps in the procedure .. do you use serum free media or media with serum...or do u use Optimem media
does serum inhibit transfection
thanks again
glad to be of help and you are most welcome..
I do my transfections in breast cancer cells and i am actually running my samples right now 
ok I basically plate in 500 ul/well. Then i make my complex in optimem media and add 100 ul of this to each well. So i basically do it in the presence of serum. I pull this off after 4-6 h and wash the wells with regular medium. Then add 1 ml of this regular serum containing medium.
One important thing is make sure that your media does not have antibiotics. If it does then transfection efficiency reduces.. Dont ask me why I have no clue...but it is..... so it is... 