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now: Sat Jun 6 12:51:20 2020 ... mod: Thu Jul 4 08:58:13 2013
With a "good" probe sequence, RadiolabelingDna by random priming is fast and convenient, however, sometimes greater sensitivity is needed. In these cases, generating a radiolabeled probe by PCR can be used. The probe has higher overall specific activity because much less unlabeled DNA template is required for PCR than for random priming. To avoid chain termination by misincorporation of unlabeled dNTPs, it is important to decrease the concentration of the unlabeled dNTPs to be similar to that of the concentration of the limiting radiolabeled dNTP (cold nucleotides each at 2x the concentration of the limiting nucleotide works well).
This protocol works well with probes from about 500 bp to 1000 bp in length.
|2.0 μl ||10x TAQ buffer with MgCl2 (NEB)|
|2.0 μl ||supercoiled plasmid DNA template at 50 pg / μl (100 pg total)|
|2.0 μl ||forward primer at 1 μM|
|2.0 μl ||reverse primer at 1 μM|
|5.0 μl ||α-32P-dATP (50 μCi @ 3000 Ci/mmol)|
|0.4 μl ||TAQ DNA polymerase at 5 U / μl (NEB)|
|2.0 μl ||dGTC-TP at 40 μM each (the other three cold nucleotides)|
|1.0 μl ||dATP at 20 μM (not radioactive → helps the synthesis reaction happen)|
|3.6 μl ||H2O|
|20 μl ||total volume|
| ||typical cycling parameters|
|3' || 94C ||initial denaturation|
|30" || 94C ||denaturation|
|30" || 55C ||anneal (may need to be optimized)|
|1' || 72C ||extension, + 2" per cycle|
| ||30 cycles|
|7' || 72C ||final extension|
| 4C ||hold|
To label DNA molecular weight markers:
Lambda phage (λ) molecular weight markers on a Southern membrane can be lit up by radiolabeling λ genomic DNA. λ digested with BstEII is available from New England Biolabs. The BstEII restriction enzyme leaves 5' overhangs that can be filled in by residual TAQ and nucleotides (either α32P-dATP or 32P-dCTP will work) after the PCR radiolabeling reaction is completed.
Denature the probe by boiling for 2' and snap cooling in an icewater bath for 2' immediately before using.