Materials and Methods

Dilute 50-500 ng of target DNA to a final volume of 5 mL with 10 mM Tris-HCl (pH 8.5), -0.1 mM EDTA and denature the DNA for 5 min at 98°C in a thermocycler with heated lid. Mix 1.5 mL probe mix (1 fmol of each probe) and 1.5 mL MLPA buffer (1.5 M KCl, 300 mM Tris-HCl pH 8.5, and 1 mM EDTA) and add to the denatured DNA. Heat the mix for 1 min at 95°C before incubating overnight (16 h) at 60°C. After hybridization dilute the sample to 40 mL with ligase mix (2.6 mM MgCl2, 5 mM Tris-HCl pH 8.5, 0.013% nonionic detergents, 0.2 mM NAD) containing 1U Ligase-65 (MRC-Holland) and perform ligation reaction of the annealed probes at 54°C for 15 min. After ligation inactivate the Ligase-65 by incubating the sample 5 min at 98°C. Mix 10 mL of the ligation mix with 30 mL of PCR buffer (20 mM Tris-HCl pH 8.5, 50 mM KCl, 1.6 mM MgCl2, and 0.01% nonionic detergents). While at 60°C, add 10 mL of a solution containing 10 pmol PCR primers, 2.5 nmol dNTPs, and 2.5 U SALSA polymerase (MRC-Holland) to the mix and perform 33 cycles of PCR (30 sec, 95°C; 30 sec, 60°C; and 1 min, 72°C) followed by a 20-min incubation at 72°C. Finally, a part of this PCR is denatured in 100% formamide, mixed with molecular

Getting Started With Dumbbells

Getting Started With Dumbbells

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.

Get My Free Ebook


Post a comment