"
Team:Freiburg/Notebook/modeling
From 2013.igem.org
Modeling Notebook
65000 cells per well were seeded. 24 h before transfection.
The cells had been transfected at 21:30 h.
 |
| Figure 1: Transfection Scheme Cells were transfected with dCas9-VP16 (transfection one) or dCas9-KRAB (transfection 3). As controll we used .... (trandfection 3 and 4). |
To generate time dependent data, one well was treated every 6 h.
t0= 01:30
t1= 07:30
t2= 13:30
t3= 19:30
t4= 01:30
t5= 07:50
t6= 13:30
Every timepoint the supernant was taken for SEAP measurement and the cells were lysated with RIPA buffer for quantitative western blotting.
The standard western blot procedure was done with 20 µl of the cell lysates.
For quantification of the western blot data, a dot blot was done. A serial dilution of anti-HA (200ng/µl)was prepared. After PVDF-membran activation and a short incubation in tranfer buffer, 2 µl of each dilution have been droped onto the membrane. After that the membrane was indubated at room temerature (1h) for drying.
The detection with the secondary anti-body was done similar to the treatment of the dCas9 blots.
