Standard Protocols

Nulcease Degradation Assay

1. Make complexes according to standard protocol
2. Add the nuclease and any co-factors that are required to the complexes. A few units of nuclease should be enough. DNase I, micrococcal nuclease and mung bean nuclease have all been used successfully.
3. Incubate at 37⁰C for around at least an hour.
4. Denature nuclease by heating the sample to 70⁰C for around 15 minutes.
5. Dissociate complexes by adding either an equal volume of trypsin (for pLL/DNA complexes) or 1.5 volume of 1% SDS and NaOH. Incubate at 37⁰C for 30 minutes.
6. Extract DNA with phenol/chloroform
7. Ethanol precipitate the DNA
8. Resuspend DNA in 20 ml TE buffer or water
9. Load onto a 0.8% agarose gel
10. Compare nuclease treated DNA with untreated plasmid DNA

This assay can be performed with fresh serum in place of pure nuclease. In this case the trypsin degradation of the complexes is preferable to the SDS treatment.

Research @ Birmingham

Gene Therapy