4.12.3. Preparation of DIG labelling (non-radioactive) probe
While DNA probes can also be used to detect RNA targets, a DIG-labeled RNA probe is ideal for detecting RNA on a Northern blot because RNA probes (riboprobes) that are transcribed in vitro are able to withstand more rigorous washing steps preventing some of the background noise. RNA probes give better sensitivity for detecting low amounts of RNA target than DNA probes. The following protocol is based on the Roche DIG RNA Labelling Kit, SP6/T7.
- Linearize the recombinant plasmid DNAs with the target insert by cutting a restriction enzyme cleavage site downstream from the cloned insert using a restriction enzyme that creates 5’ overhangs (the choice of this enzyme will depend on the sequence of both the plasmid and insert).
- After restriction digestion, purify the DNA by
spin column purification or via phenol/chloroform/isoamyl alcohol extraction
This is commonly referred to as the ‘plasmid mini-prep’ and there are numerous commercial and home-made recipes for doing so that all work well.
- Add the
2 μg purified template DNA to the following transcription reaction mixture to
make 26 ml probe as
4 ml 10X NTP labelling mixture,
4 ml 10X Transcription buffer,
2 ml Protector RNase Inhibitor,
4 ml RNA Polymerase SP6/or T7.
Adjust the volume with additional water until a final volume of 26 ml.
transcription reaction in the 37°C incubator for 2 hours.
Longer incubations do not increase the yield of labeled RNA.
Stop reaction with 2μl 0.2M EDTA (pH 8).
Labeled probes are stable for at least one year at -15 to -25°C.