Hybridization Conditions

Northern hybridization conditions for most probes greater than 50 bases in length can be very closely approximated using the following formulas:

For RNA probes:

Use of formamide reduces the Tm by: Tm - 0.35°C (%formamide)

Tm = 78°C
   +  16.6 [(log10)([Na⁺/1.0+0.7[Na⁺])]
   +   0.7 (%G+C)
   - 500/duplex length
   -   1°C per %mismatch

For DNA probes:

Use of formamide reduces the Tm by: Tm - 0.50°C (%formamide)

Tm = 76°C
   +  16.6 [(log10)([Na⁺/1.0+0.7[Na⁺])]
   +   0.8 (%G+C)
   - 500/duplex length (for random-primed probes, use length of average probe molecule, about 200-300 bases).
   -   1°C per %mismatch

For oligonucleotides:

Effects of mismatches in short duplex regions can be profoundly influenced by context and position effects. For example, mismatches in the center of a duplex will reduce the Tm much more than a mismatch at the end.

   1-1.5°C per %mismatch

Note: Final probe concentration should be between 1-10 ng/ml or 0.1-2 pmoles/ml (1 x 10⁶ cpm/ml of a probe with a specific activity of 1 x 10⁹ cpm/µg is equal to 1 ng/ml).

"Tm" is the temprature, at equilibrium in a given solution, at which 50% of the base pairs are in a duplex are paired. To drive the hybridization towards completion and speed hybridization, RNA and DNA probes are usually hybridized 15-25°C below the actual Tm, and oligonucleotides are hybridized 5-10°C below the actual Tm. A range of optimal hybridization temperatures must be given because there is no way to predict how many inappropriate sequences any particular probe will bind to. Actual hybridization should be carried out at the lowest temperature that gives little or no cross ybridization to other sequences. A pilot experiment should be done to determine actual hybridization temperatures from within this range. These calculations illustrate some important factors:

The effect of the probe length is rather small.

The effect of G+C content can be substantial.

Using an RNA probe in a Northern instead of a DNA probe results in an increase in Tm of 8-18°C (13°C with G+C = 50%)