Standard Protocols

Gel Retardation assay for complex formation

The condensation of DNA by cationic polymers can be measured by a reduction of the mobility of DNA on agarose that leads to the retention of DNA in the wells.

1. Place calf thymus or plasmid DNA (20 µg/ml) in a microcentrifuge tubes containing 1 ml of water.
2. Add cationic polymer to the microcentrifuge tubes to form complexes at different charge ratios.
3. Incubated at room temperature for 30 minutes to allow the complexes to form properly.
4. After this time place 20 µl aliquots of the complexes in fresh microcentrifuge tubes containing 2 µl of gel loading buffer.
5. Load the complexes onto a 0.8 % agarose gel in TBE buffer containing ethidium bromide.
6. Electrophorese the gel in TBE buffer at 90V for 60 minutes.
7. Visualise and photograph the gel on a UV transilluminator.

This assay can be performed using radiolabelled DNA and analysed on the PhoshorImager to quantify how much DNA is retained in the wells at different charge ratios.

Research @ Birmingham

Gene Therapy