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Amount of Probe to Use in a Northern Blot

With ULTRAhyb™:

Probe Type Final Concentration in Hybridization Buffer

Radiolabeled RNA and DNA* probes

 106 cpm/ml
Nonisotopic DNA* probes labeled by enzymatic incorporation of a nonisotopically modified nucleotide Up to 10 pM
Nonisotopic DNA* probes labeled by chemical labeling method (e.g., Ambion's BrightStar™ Psoralen-Biotin 1 pM
Nonisotopic RNA probe 0.1 nM
Oligonucleotide probes Not recommended (use ULTRAhyb™-Oligo)
*Be sure to denature dsDNA probes prior to hybridization.

 

With ULTRAhyb™-Oligo:

Probe Type Final Concentration in Hybridization Buffer

Radiolabeled DNA oligonucleotide

106 cpm/ml

Recommended Oligonucleotide Probe Characteristics:

Characteristic Recommendation

Length

 26-45
Tm value* 78-90¨C
GC content 47-62%
Specific activity 5 x 106 cpm/pmol
Labeling method 5' end labeling with [gamma-32P] ATP
*DNA oligonucleotides with a Tm outside of the range listed may require a different hybridization temperature that would have to be determined empirically.

 

With Most Other Hybridization Solutions:

Probe Type Final Concentration in Hybridization Buffer

Radiolabeled RNA and DNA* probes

 1 x 106 to 5 x 106 cpm/ml
Nonisotopic RNA or DNA* probes 0.1 nM
Oligonucleotide probes** 1 x 106 to 5 x 106 cpm/ml
*Be sure to denature dsDNA probes prior to hybridization.
**Note: Not all hybridization solutions are compatible with oligonucleotide probes.

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