CaCl2 transfection of cells in culture
1. Plate cells and let them adhere for 6h (Or O.N). The cells should reach
70% density.
2. In eppendorf tubes mix transfection solution without 2xHBS:
For a 10cm petri dish:
500μl DDW + plasmids (0.5μg OT/OF, 0.05μg β-Gal)
60μl [2M] CaCl2
500μl 2xHBS buffer
Total volume: 1ml
For a 6-well plate:
100μl DDW + plasmids (0.5μg OT/OF, 0.05μg β-Gal)
12μl [2M] CaCl2
100μl 2xHBS buffer
Total volume: 200μl/ per well
For a 24-well plate:
50μl DDW + plasmids (0.5μg OT/OF, 0.05μg β-Gal)
6μl [2M] CaCl2
50μl 2xHBS buffer
Total volume: 100μl/ per well
3. Dived the transfection mix into 2 eppendorf tubes:
#1 Add 0.5ug empty GFP plasmid
#2 Add 0.5ug β-catenin plasmid
4. Add the 2x HBS drop by drop and vortex.
5. Incubate at R.T for 20-30 min.
6. Vortex and add the transfection solution to the plated cells drop by drop
into the growth medium.
7. Incubate for 24h at 37C.
8. Change to a fresh medium containing activation protein.
9. Incubate for 24h at 37C.
10. Discard medium.
11. Wash 1xPBS.
12. Discard PBS and add 100μl lysis buffer + protease inhibitor.
13. Incubate 10min on ice.
14. Transfer the lysate to a fresh eppendorf tube.
15. Incubate 10min on ice.
16. Centrifuge for 15min, 13,000 rpm at 4C.
17. Perform Luciferase and β-Gal assays.
18. Luciferase assay: Add 50μl of luciferin buffer to 10μl of lysate supernatant
and read immediately.
19. β-Gal assay: In a 96-well plate- Put 10μl of lysate supernatant, add 149μl
ONPG buffer. Incubate at 37C for a few minutes or at R.T until color is
develop. Read at 420nm.
Luciferase assay
1. Luciferin: 100mg in 3.57ml 10mM Tris-Acetate (pH 7.8), 100μl aliquots at 20C.
2. ATP: 200mM in ??? DDW, 150μl aliquots
3. Luciferase buffer: 100mM Tris-Acetate (pH 7.8)
100mM MgAc
1mM EDTA
(Tris-Acetate: 1M Tris adjusted to pH 7.8 with Acetic acid)
For 500ml: 50ml Tris 1M
5ml MgAc 1M
1ml EDTA 0.5M
444ml DDW
100μl luciferin + 150μl ATP in 13.5ml luciferase buffer → 50μl per reaction
For the reading: Add 50μl of luciferin buffer to 10μl of lysate supernatant
and read immediately.
β-Gal assay
For each reaction:
30μl ONPG
1.5μl MgCl2 x100
115μl PBSx1
Total volume: 149μl
In a 96-well plate:
10μl protein extract (lysate)
149μl ONPG mix
Incubate at 37C for a few minutes or at R.T until color is develop. Read at 420nm.
MgCl2 x100:
500μl MgCl2 [1M]
1.54ml β-MER
2.96ml DDW
Total volume: 5ml
ONPG: 4mg ONPG in 1ml PBSx1