promega雙熒光素酶報告基因檢測系統(tǒng)快速Protocol

1、promega雙熒光素酶報告基因檢測系統(tǒng)快速protocol?ordering /technical information:reagent preparation1.1x p l b :add 1 volume of 55x p a s s i v e l y s i s b u f f e r (p p l b ) to 4 volumes of distilled water. mix well. store at 4c (1 month).2.l a r i i : resuspend the lyophilized ll u c i f e r a s e a s s a y s u

2、 b s t r a t e in l l u c i f e r a s e a s s a y b u f f e r i i (10ml for cat.# e1910, e1960; 105ml for cat.# e1980). store at 20c (1 month) or 70c (1 year).3.s t o p & g l o ?r e a g e n t :a.add 2.1ml of 50x ss t o p & g l o ?s u b s t r a t e to 105ml of s s t o p & g l o ?b u f f e r in the am

3、ber s s t o p & g l o ? r e a g e n t bottle provided. vortex 10 seconds. store at 20c for 15 days.b.for a smaller amount of 11x s t o p & g l o ? r e a g e n t :to the required amount of s s t o p & g l o ?b u f f e r , add 50x s t o p & g l o ?s u b s t r a t e to a final 1x concentration. (for ex

4、ample, add 0.2ml of 50x ss t o p & g l o ?s u b s t r a t e to 10ml of ss t o p & g l o ?b u f f e r to make a 1x solution of s s t o p & g l o ? r e a g e n t .)cell lysis1.remove growth media from cultured cells.2.rinse cultured cells in 1x pbs. remove all rinse solution.3.dispense the recommended

5、 volume (below) of 11x p l b into each culture vessel.volumes of 1x plb to use in step 3.passive lysis active lysis plate size 1x plb dish/plate size 1x plb 6-well 500l 100 200mm 1ml 12-well 250l 60 15mm 400l 24-well 100l 35 12mm 200l 48-well 65l 6-well 250l 96-well 20l 12-well 100l 4.passive lysis

6、:gently rock/shake the culture vessel for 15 minutes at room temperature. transfer lysate to a tube or vial.*for automated applications, the dlr? assay is performed directly in the multiwell plate.additional protocol information is available in technical manual #tm040 or #tm046, available online at:

7、cell lysisremove growth media from cells.rinse with 1x pbs.add 11x p l b .perform lysis.transfer to a new tube or vial.*2866m a 02_0a ? ordering /technical information:dual-luciferase ?and dual-luciferase ? 1000 assay protocolsadditional protocol information is available in technical manual #tm040 o

8、r #tm046, available online at:plate with 20l of plb lysate/well.dispense 100l of l a r i i .measure firefly luciferase activity.dispense 100l of s t o p & g l o ?r e a g e n t .measure renilla luciferase activity.assay with manual or single-injector luminometerpredispense100l of ll a r i i into lumi

9、nometer tube.program luminometer.transfer 20l of plb lysate. mix.measure firefly luciferase activity.dispense 100l of s t o p & g l o ?r e a g e n t .measure renilla luciferase activity.assay with 96-well platebefore you begin:set injectors 1 and 2 to dispense 100l of l a r i i and s s t o p & g l o ?r e a g e n t , respectively.for measurements, use a 1-