Hybridization temperature optimization

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. 

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. 

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. 

Temperature Elevated temperature improves hybridization efficiency by speeding diffusion and reducing secondary structure, however excessive temperature reduces the efficiency by melting duplex Optimal hybridization temperature is 10°C below the melting temperature of the heteroduplex. 

Sequence Detector System and heated to 90 C for 5 minutes. The temperature was then reduced at 1°C per minute increments to 20°C. Data were recorded at each temperature interval. The optimal hybridization temperatures for each beacon were determined from these plots. 

Fig. 14 Optimization of hybridization conditions, (A) Plot of peak current response vs. incubation time (t from 10 min to 40 min), (B) Effect of hybridization temperature (from 40°Cto 70 °C), The differential pulse voltammetry peak current of CA-MB was detected in 0,1 M phosphate buffer saline solution pH 7,3, Reproduced from Li with permission... 

Despite the decrease in annealing rate, formamide presents unique advantages in nucleic acid hybridization. It is used to lower the hybridization temperature and to reduce the risk of thermal strand scissions or to control the stringency of annealing (36). A 30—50% formamide concentration allows optimal hybridization to be performed at 30—42°C rather than at 68°C without formamide. 

This process has many benefits in the context of green chemistry it involves two enzymatic steps, in a one-pot procedure, in water as solvent at ambient temperature. It has one shortcoming, however-penicillin acylase generally works well only with amines containing an aromatic moiety and poor enantioselectivities are often observed with simple aliphatic amines. Hence, for the easy-on/easy-off resolution of aliphatic amines a hybrid form was developed in which a hpase [Candida antarctica hpase B (CALB)] was used for the acylation step and peniciUin acylase for the deacylahon step [22]. The structure of the acyl donor was also optimized to combine a high enanhoselectivity in the first step with facile deacylation in the second step. It was found that pyridyl-3-acetic acid esters gave optimum results (see Scheme 6.8). 

The first step of a PCR involves DNA denaturation at 90-95 °C, in a buffered, neutral, aqueous solution containing DNA polymerase, the four deoxynucleotide triphosphates and Mg++, in the presence of a large excess of the two primers (Fig. 27). In the second step, the temperature of the reaction is lowered to about 10 °C below the melting temperature of the primers and the primers (which are considerably smaller than the DNA) are allowed to hybridize to their complementary sequence on the DNA template molecule. This temperature is still too high for the DNA to fully renature. The temperature is then raised to 72 °C, the optimal temperature for extension of the primers by the DNA polymerase, which catalyses the addition of nucleotide triphosphates to extend the sequence in each direction from the... 

Urea (0.01 M), sodium carbonate (0.01 M), magnesium chloride (0.01 M), or distilled water can be used as microwave fluids to obtain similar results in terms of both sensitivity and intensity of the hybridization signal. Alternatively, 10 mM citrate buffer (pH 6.0) can be used as the microwave fluid. The major role of these fluids is to mediate high temperature effects, which is confirmed by the achievement of a good hybridization signal using distilled water. Note that pretreatment conditions must be optimized for every tissue type and for every cell type in a given section. 

Phosphoramidites, a ligand class that has only recently been introduced into asymmetric hydrogenation, in the form of hybrid chelate ligands [29], induce excellent enantioselectivity as monodentate ligands. Thus de Vries, Feringa, and co-workers could reduce standard substrates in >96% ee with a rhodium complex based upon the binaphtholphosphoramidite 3d, once the solvent and reaction temperature had been optimized [30],... 

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