Transfection normailsing using dot-blot hybridisation
Contributed by Alexei Gratchev
It is a known, that the usage of plasmid expressing reporter gene driven by strong promoter causes problems when one wants to compare different cell lines. This method allowes you to measure directly the amount of the plasmid which entered the cells and therefore provides a reliable reference.
- Harvest your cells after transfection and transfer 60mkl of the suspension to a new tube.
- Add proteinase K to the final concentration 0.4 mg/ml, incubate 2-12 h at 65° C.
- Add 200 mkl phenol:chlorophorm (1:1) mixture, vortex vigorously, and centrifuge 1 min at 13000 rpm.
- Transfer the aqeous phase to a new tube
- Add 200 mkl chlorophorm, vortex and centrifuge 1 min at 13000 rpm.
- Transfer 40 mkl of lisate to a new tube and add 160 mkl 0.5 M NaOH.
- Denature DNA by heating the sample 10 min at 80° C.
- Blot obtained sample on the membrane using any dot blotting instrument.
- Neutralise the membrane in 2xSSC.
- Fix DNA on the membrane by backing it for 2 h at 80° C.
- Hybridise the membrane with reporter gene or plasmid vector probe using standard Southern protocol.
- Analyse obtained autoradiogramm densitometrically.
- Optional: in the case of too strong signals or big range of signal intensity it is usefull to cut out hybridised spots and analyse amount of radioactivity using scintillation counter.