Transfection normalizing using dot-blot hybridisation

Contributed by Dr. Alexei Gratchev

It is known, that the usage of plasmid expressing reporter gene driven by strong promoter as an internal transfection control causes problems when one wants to compare different cell lines.

Method described here allowes you to measure directly the amount of the plasmid that entered the cells and, therefore, provides a reliable reference. The details may be found in Eur J Cell Biol. 2000, 79(2):150-3.

  1. Harvest your cells after transfection and transfer 60mkl of the suspension to a new tube.
  2. Add proteinase K to the final concentration 0.4 mg/ml, incubate 2-12 h at 65° C.
  3. Add 200 mkl phenol:chlorophorm (1:1) mixture, vortex vigorously, and centrifuge 1 min at 13000 rpm.
  4. Transfer the aqeous phase to a new tube
  5. Add 200 mkl chlorophorm, vortex and centrifuge 1 min at 13000 rpm.
  6. Transfer 40 mkl of lisate to a new tube and add 160 mkl 0.5 M NaOH.
  7. Denature DNA by heating the sample 10 min at 80° C.
  8. Blot obtained sample on the membrane using any dot blotting instrument.
  9. Neutralise the membrane in 2xSSC.
  10. Fix DNA on the membrane by backing it for 2 h at 80° C.
  11. Hybridise the membrane with reporter gene or plasmid vector probe using standard Southern protocol.
  12. Analyse obtained autoradiogramm densitometrically.
  13. 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.

Recommended reading

  1. Siedow A, Gratchev A, Hanski C. Correct evaluation of reporter assays in different cell lines by direct determination of the introduced plasmid amount. Eur J Cell Biol 2000; 79(2):150-153.


\METHODS\Transfection\Transfection normalisation using dot blot