3.3.3. Synthesis of DIG-labelled DNA probe
DIG-labelled probes offer a method to identify where probes have attached on the membrane (e.g.., the location of their targeted DNA match). These DIG probes are an alternative to highly regulated and more dangerous radio isotopic probes.
- Mix the DIG-labelled PCR reaction components
from the Roche Applied Science PCR
DIG Labelling Mix with the probe template as follows:
5 ml PCR Buffer (10X),
5 ml PCR DIG Labelling Mix,
0.5 ml Upstream Primer (25 mM),
0.5 ml Downstream Primer (25 mM),
0.5-1 ml Template (plasmid DNA, 10-100 pg, or genomic DNA, 1-50 ng),
0.75 ml Enzyme Mix,
Add sterile water until total reaction volume is equal to 50 ml.
- Set the annealing temperature of PCR reaction to reflect the predicted annealing temperature of the primers, also reported at the time of purchase.
- The kit contains a post-hoc check for probe labelling efficiency that is recommended.