Digoxigenin (DIG)-Labelling DNA for use as Hybridization Probes† Protocol: 1. Set up the following 50µl PCR reaction on ice... Mixer Volume H2O 32.75 µl 10x ExTaq Buffer 5 µl ExTaq dNTP's 4µl DMSO 5 µl 1 nmol/µl DIG-11-UTP (Roche #1-093-088) 0.5 µl 0.5 µg/µl Primer 1 (63<Tm<70) 0.5 µl 0.5 µg/µl Primer 2 (63<Tm<70) 0.5 µl DNA Template (1ng/µl gel purified fragment or plasmid) 1 µl 1 unit/µl Taq Polymerase 1 µl 2) Thermocycle as follows being sure to add the reaction to the thermocycler once the block has reached 94°C... Step Repeats Temp (°C) Time 1 1x 94 2min. 2 35x 94 62 72 45s 1min 2min 3 1x 72 5min 3) Run 20 µl of the products on an Agarose gel. The DIG-labelled product should be a bit larger than the control PCR product lacking DIG. Cut out the dig and control bands. Gene clean both of these bands and run out 1-3ul on a 1.0% agarose gel with a DNA Mass Ladder to estimate concentration. Use 100-200ng of labelled probe in Southern Blot Hybridizations. Each batch of dilute probe can be used for multiple rounds of hybridization (stored at –20¾).   DNA Template: The template used for making the DIG probe can be either a plasmid containing the gene of interest or a gel purified PCR product. The only stipulation is that the identity of the PCR product or plasmid template used for probe preparation be confirmed by DNA sequencing.   †Notes: If you found your way to this page because you're interested in using DIG as a non-radioactive probe for anything other than the Photosynthetic Mutant Library, you may want to check Roche's website...they have a 200+ page manual on DIG labeling. The ratio of dTTP to DIG-11-UTP we're using is 1:20, this generates probes that are of sufficient specific activity for probing PCR products by hybridization. However, a 1:3 ratio is more appropriate if you intend using the probe on a genomic Southern (page 183-186 of the Roche DIG manual).

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