Twenty microliters of post-PCR mixture is denatured by the addition of 180 mL of 0.4 M NaOH, 20 mM EDTA, which may include 4 mL of 0.02% Orange II (optional spotting dye used to trace the blotting accuracy). Equal portions of the resulting solution are spotted in parallel on two previously water-wet Hybond N+ nylon membranes (Amersham Pharmacia Biotech), thus creating two exact dot blot replicates of the analyzed DNA segments. Manifold is usually used to transfer DNA onto the membrane but direct spotting can also be used. DNA is then fixed on the membranes by UV irradiation (e.g., using light of 254 nm at 120,000 mJ/cm2 in Stratalinker 1800; Stratagene) and the membrane is subsequently washed to remove NaOH and excess salt.
1. It is known that both strands of the DNA are not necessarily equivalent as probes in hybridization. This effect was observed with longer probes but also applies to short oligonucleotides. Purine-rich oligo-nucleotides have a tendency to be better binders to a longer template than their pyrimidine-rich equiva-lents. The former would maintain a more rigid structure, stabilized by stacking interactions because of the presence of purines, thus favoring template binding. The latter that are more coiled need additional energy to adopt helical extended structure consistent with double-stranded duplex formation. Note that pyrimidine-rich complement within a longer template is stabilized by stacking with its nearest neighbors from 5' and 3' sides through a danglingends effect, such that it is less coiled than its isolated/ naked oligonucleotide complement. In conclusion, choosing (+) rather than (—) strand for ASO design may modify the kinetics of duplex formation and its stability, and can be used to improve the test. 2. Although the rule of thumb is to place the probed polymorphic site in the middle of ASO, it is advisable to readjust its position if both flanking sequences differ in stability because of uneven composition in (G+C)/(A+T) and/or unequal redistribution of py-rimidines and purines.
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The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.