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now: Thu Jun 24 08:18:09 2021 ... mod: Wed Mar 31 17:28:49 2021
Radiolabeling DNA for use as hybridization probes
We use Stratagene's PrimeiIt II kit (http://www.stratagene.com/products/showProduct.aspx?pid=117
) and mostly follow the instructions. This kit uses random nonamer primers that are extended by Klenow polymerase. The random nonamers work significantly better than random hexamers found in other radiolabeling kits.
If the probe sensitivity by random priming is not adequate, PcrRadiolabeling is an alternative approach that may work better.
preparation: you will need a boiling water bath, a bucket of ice with water in the bottom, and a heat block at 37C with water in one of the heat block wells to assure good thermal contact
- start with ~25 ng of DNA to be labeled in an eppendorf tube
- If you want to also light up a lambda ladder add 10 picograms of lambda DNA per lane of lambda run on the gel to be probed.
- DNA templates between 400 bp to 1000 bp seem to work the best
- in general using more DNA than 25 ng is not better and is sometimes worse due to high background
- add water to get a total volume of all DNA to be labeled plus water of 24 μl (25 μl if using 6000 Ci/mmol 32P)
- add 10 μl of random nonamer primer solution. mix well
- put an eppendorf lid clamp on the tube and incubate in a boiling water bath for 2 minutes, then immediately place in an ice/water bath for 2 minutes, then briefly centrifuge to collect any condensate and immediately place back on ice.
- add 10 μl of the appropriate 5x primer buffer and mix by pipetting up and down
- there are separate primer buffers for using α-32P-dATP and α-32P-dCTP. be certain you use the correct one
- add 1 μl of Exo- Klenow enzyme (5U) into the tube on ice, but do not mix
- add 5 μl of the appropriate radionucleotide (such as 3000 Ci/mmol α-32P-dATP), mix thoroughly by pipetting up and down and incubate at 37 C for 10 minutes
- if using 6000 Ci/mmol 32P use 4 μl rather than 5 μl, this way you get three complete reactions from a 250 μCi 32P vial (12.5 μl). The reaction works just fine with 4 μl instead of 5 μl
- be certain to use α-labeled 32P (rather than γ-labeled), and that you are using a dNTP rather than an rNTP and that your labeling buffer matches the flavor of radiolabed dNTP you have
- add 2 μl of stop solution and mix well
Clean up and quantify your radiolabeled probe.
Denature the probe by boiling for 2' and snap cooling in an icewater bath for 2' immediately before using.