Probe hybridization temperature

Hybridize sample at 42° overnight (preferably in an oven to eliminate condensation). The optimal hybridization temperature must be empirically determined and is usually between 40 and 60° however, most probes will hybridize at 42 to 45°. 

The success of hybridization often depends on several factors that affect the rate of probe binding to the target DNA on the membrane. The most important ones include hybridization temperature, probe concentration, ionic strength, pH, and viscosity of hybridization solution (Wahl, 1987). As the hybridization temperature is one of the crucial parameters, its optimization has to be considered. Equation (4.1) is often used for estimating hybridization temperature, where n is the number of nucleotides in the probe and the Na+ concentration of the hybridization buffer is 1 m or less. 

After Southern transfer of restriction-digested genomic DNA, filters are prehybridized for 1-4 hr. The probe is boiled for 10 min to denature it, added to fresh hybridization buffer, and hybridized overnight. To calculate appropriate hybridization temperatures in buffers with or without forma-mide, and for details of hybridization buffers and washes, see standard protocols.13 For hybridizations at low stringency to detect signals from fragments of approximately 65-70% overall sequence similarity, we apply 55° for hybridization and washes in 6X SSC without formamide. Similar protocols can be used for colony and plaque hybridizations. 

Denaturation of double-stranded target and probe Elevated temperature and/or alkaline treatment is most often used to render the double-stranded (DNA) probes and targets single-stranded as a prelude to hybridization. 

Denature DNA of chromosomes by temperature and salt adjustment Add labeled probe at optimum concentration and at conditions optimum for hybridization (temperature, salt concentration), use optimum hybridization time Remove unhybridized probe by washing Process for visualization of hybridization... 

Concentrations of oligonucleotide probes and antibody, hybridization temperature, and incubation times correspond to those used in the authors laboratory. However, it may be necessary to optimize each of these parameters, with those given in the protocol serving as a good starting point. For oligonucleotide probes, the authors have found 1 pmol of each as a universally suitable concentration. 

Gently heat cells to 60-70°C to denature DNA, add probe and incubate at the hybridization temperature (usually room temperature). 

The hybridization buffer usually contains 5 X SSC, 50% formamide, 100 xg/ml carrier DNA/tRNA, 0.1% Triton X-100 and 20 mM vanadyl ribonucleoside in addition to denatured probe. In the case of optimal probes of 150 bases (40-60% GC), the optimal hybridization temperature (T — 25 0 in this buffer would be about 56-70°C for RNA probes and 37-6 TC for DNA probes (eqs. (11) and (12), Chapter 2). However, very high temperatures, e.g., > 50°C, affect tissue quality. Usually, incubation is at 20-50°C in a humidified environment. 

Carry out hybridization overnight under RNase-free coverslips (avoid trapping air bubbles under the coverslip ) with 20 ng of probe per slide (in 80-pL hybridization buffer see Section 3.1., item 6) at the optimum hybridization temperature (T[ ), calculated for each probe (see Notes 13-15). 

Hybridize the section for 16-20 h see Note 4) at suitable temperature in a sealed humid chamber containing 5X SSC. A different hybridization temperature will be needed for various probes according to their see Note 5). 

The hybridization temperature is dependent on the type of probes that have been used. With hybridization buffer containing 50% formamide, it is suggested that the following range of temperature should be tested initially 42-48°C for cDNA probes 42-55°C for cRNA probes 37-40°C for oligonucleotide probes. 

Hybridization with probes Hybridization must be carried out under stringent conditions to ensure specific annealing of the probe to the target sequence. Stringency is adjusted by varying either the formamide concentration in the hybridization buffer or the hybridization temperature. The hybridization takes place by applying the fluorescently labeled probes to the samples in the presence of a hybridization buffer. Hybridization should be performed in a dark humid chamber usually at temperatures ranging from 27 to 50 °C. 

The hybridization temperature may need to be altered. Oligonucleotide probes are hybridized at 30-37°C overnight (4,5). The cRNA probes are hybridized at40-70°C (3,5). Also bubbles may form under the coverslip during hybridization. This can be avoided by degassing the hybridization solution for 30 min under vacuum prior to use. 

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