DR-GFP reporter assay

Hi Yagiz,

Thanks for your query. Here is the information on DR-GFP plasmid that is also given in our paper and also here for your convinience  https://www.addgene.org/2647 .This plasmid comes from the study https://genesdev.cshlp.org/content/13/20/2633

Here also attaching the detailed protocol that was used.

Hope these would be helpful for your own study.

All the best,

Nitika Taneja

DR-GFP Double Transfection Protocol 

Reagents:

1. Xtremegene reagent
2. Opti-MEM
3. Complete medium
4. Lipofatamine RNAiMAX
5. siRNA (si-control/si-BRCA1)
6. DNA plasmid (Scam, P-MAX, DR-GFP, I-SEC1)

Experimental 6-well plate design:

Protocol:
 

Day 0

Seed 1*10^5 /2ml cells per well in 6 well plate

Day 1 (siRNA transfection):
Add 150µl Warm OptiMEM + 6µl Lipofectamine RNAiMAX Reagent in an 1.5ml Eppendorf (A), vortex 

1. Add 150µl Warm OptiMEM+ 4µl siBRCA1/ siNT in another 1.5ml Eppendorf (B), vortex
2. Transfer content of Eppendorf (B) into Eppendorf (A) in 1:1 ration, vortex and incubate for  15 mins @RT 
3. Meanwhile change the media into new complete 1.7ml media to in a 6well dish/ 60mm dish
4. Add 300µl of the sample mix dropwise and shake

Day2 (siRNA transfection):

1. Add 150µl Warm OptiMEM + 6µl Lipofectamine RNAiMAX Reagent in an 1.5ml Eppendorf (A), vortex 
2. Add 150µl Warm OptiMEM+ 4µl siBRCA1/ siNT in another 1.5ml Eppendorf (B), vortex
3. Transfer content of Eppendorf (B) into Eppendorf (A) in 1:1 ration, vortex and incubate for  15 mins @RT 
4. Meanwhile change the media into new complete 1.7ml media to in a 6well dish/ 60mm dish
5. Add 300µl of the sample mix dropwise and shake

Day3 (plasmid transfection):

Prepare the medium

1. Add 100µl Warm Opti-MEM medium in an 1.5ml Eppendorf
2. Add  6µl Xtremegene in the centre
   (DO NOT PIPETTE UP AND DOWN), 
   tap and vortex and incubate for 5 mins, 
3. Add 3µg of the DNA content (1.5ug of each plasmid)
   (DO NOT PIPETTE UP AND DOWN)
   tap and vortex and incubate for 15 mins
    
4. Add the transfection mix dropwise and evenly in the new medium on the dish
5. Rotate to mix well 

Day 4 (plasmid transfection) (some samples may need to tryspinise and expand):
Replace the culture medium with 1.5ml medium

1. Add 100µl Warm Opti-MEM medium in an 1.5ml Eppendorf
2. Add  6µl Xtremegene in the centre
   (DO NOT PIPETTE UP AND DOWN), 
   tap and vortex and incubate for 5 mins, 
3. Add 3µg of the DNA content (1.5ug of each plasmid)
   (DO NOT PIPETTE UP AND DOWN)
    
4. Add the transfection mix dropwise and evenly in the new medium on the dish
5. Rotate to mix well 

Day 5: FACS analysis

Trypsinise the cells, wash with PBS and resuspend in 300-500µl PBS, keep it on ice before analysis