Prehybridization conditions

As a general rule, prehybridization conditions (buffer and temperature) are, except for ohgonucleotide probes and charged nylon, identical to those of hybridization although the time is shorter (usually fast but prolonged prehybridization does not harm). Most (pre)hybridization buffers are variants of two basic buffers, one with formamide and one without. Formamide allows the use of lower incubation temperatures and an easy adjustment of the stringency and is generally used, or even essential, for RNA hybridization or for nonradioactive probes. It is recommended, particularly when formamide is used, to include a buffer, such as sodium/potassium phosphate, Tris-HCl, or PIPES-NaOH, or to use SSPE instead of... 

Blots are hybridized under the same conditions as for prehybridization with the addition of denatured labeled probe. The labeled probe has a specific activity of at least 0.5 X 108 counts/min (cpm)//ig and is present at a concentration of at least 1 X 106 cpm/ml. The probe is denatured by treatment with 1/100 volume of 10 ANaOH for 10 min at 68° followed by neutralization with 1/10 volume of untitrated 1.5 M NaH2P04. The blots are hybridized for 40-48 hr at 37° between glass plates in a water bath. 

Five steps can be distinguished in membrane hybridization (i) immobilization of target nucleic acid (ii) prehybridization to saturate the remaining binding sites which would otherwise adsorb probe non-specifically (iii) hybridization in low stringent conditions to adsorb probe as efficiently as possible (iv) posthybridization washes to define the stringency of the hybridization and thus the specificity of the reaction (v) the detection step (Fig. 8.2). In addition, the hybridized probes can sometimes be stripped from the blots to expose the targets to other probes. 

The following conditions work well with Hybond N and N (Amersham) membranes using a prehybridization/hybridization buffer made according to the following recipe. For 50 ml of buffer, combine 40 ml sterile distilled... 

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