quick question - b-gal assay (Jun/30/2008 )

Hi,
I¡m using Bgal plasmid as control in transfection with luciferase reporter assay.
B gal activity is then measured by spectrophotometry; this is the protocol:

Spin down the cell lysate.
Put 50 ul of cell lysate supernatant into a microcentrifuge tube.
Add 500 ul of Z buffer with b-2 ME.
Add 100 ul of ONPG (4 mg/ml) and Vortex.
Incubate the tubes at 30 C till you see yellow color.
Stop the reaction by add 250 ul of 1 M Na2CO3
Read at OD=420 (visible light).

DOES ANYONE KNOW FOR HOW LONG CAN I LEAVE THE SAMPLES, IF YELLOW COLOUR DOESN'T APPEAR AFTER HALF AN HOUR?
AND IF YELLOW COLOUR APPEARS CAN I STOPPED THE REACTION AND SAVE THE SAMPLES TO MEASURE ON THE FOLLOWING DAY.??


THANK YOU

BIOTECH


Reagent
Z Buffer: Per liter
16.1 g Na2HPO4.7H2O
5.5 g NaH2PO4.H2O
0.75 g Kcl
0.246 g MgSO4.7H2O
Z buffer with b-2ME : mix 27 ul of b-2ME with 10 ml of Z buffer.
ONPG (O-Nitrophenyl-b-D-galactopyranoside): 4 mg/ml in H2O
1 M Na2CO3
Procedure

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I have done upto 2 hrs. I assume longer time is possible if you have a control with untransfected cell lysate for background deduction.

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THANKS!

I THINK SO TOO.......BUT WHO KNOWS IF STABILITY OR OTHER THINGS MAY AFFECT IT, i'VE DONE THE ASSAY FOR THE FIRST TIME AND SAMPLES DIDN'T TURN YELLOW, I REPEATED IT WITH MORE LYSATE AND MORE ONPG (70UL LYSATE AND 150/200UL ONPG), AND LEAVED IT FOR A WHILE, AS IT DIDN'T CHANGE THE COLOUR IN A FEW HOURS I LEFT IT ON AND GUESS WHAT.......NOW THERE IS A LIGHT COLOUR IN THE TUBES!!! DIFFERENT FROM THE CONTROL....SO THE IDEA IS TO DO IT AGAIN AND MEASURE IT.

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The best way is to improve your transfection efficiency. :-))

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YES MAYBE IT WAS BECAUSE OF THAT,

I USED 1,5 UG DNA /WELL OF HELA CELLS (6 WELL PLATES)..........THE RATIO WAS 1:1 THIS IS 0,75UG B-GAL PLASMID AND 0,75UG PGL3 (THE PLASMID WITH THE CONSTRUCTION I'M STUDYING); USED LIPOFECTAMINE 2000 AND ASSAYED THE CELLS 24HS AFTER TRANSFECTION.

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Ranilla Luciferase is the way to go..

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biotech!:

you can adjust the ratio of b-gal construct to luciferase construct to 2:1 w/w.

I normally do 96 well plate transfection for this type of experiements (0.2-0.3 ug per well 1:4-1:5 ratio of DNA to lipo2000, 100 ul final volume with 5% FBS during transfection), but I think with some optimization, you should be able to get decent results using 48 well.

You save a lot of reagents and DNA and you can include a lot more replication sample per experiment.




ellcunter: yes, if the cost is not an issue, this is the simplist way.

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