Aging Aging

106 Engel, Adibzadeh, and Pawelecamplification might contain some fragments of the same size, but with differentsequences. This is based on the randomly amplified products in the PCR,which can result in oligonucleotides coincidentally of the same size. The differentialexpression of the DNA fragments has to be confirmed, i.e., by Northernblotting (see also Note 5).3.6. DNA Gel Electrophoresis1. Agarose gels are prepared with 1× TBE buffer and should have a percentage of1.5–2.0% agarose.2. DNA gel electrophoresis is done in a horizontal manner. The gels have to becovered with running buffer (1× TBE). DNA probes are diluted 1:5 (v/v) insample buffer and loaded into the sample wells of the gels (depending on theconcentration and the volume of the gel wells, 10–40 µL of the DNA probe canbe taken). To determine the size of the products it would again be useful to applyadditionally a molecular weight marker. The orientation of the electrodes has tobe chosen so that the DNA can run in the direction of the anode (positivelycharged electrode), because DNA is negatively charged.The voltage should be set to 1–15 V/cm (distance between the electrodes). Thetracking dyes incorporated into the sample buffer serve as markers for theprogress of the run.3. At the end, the DNA is stained with ethidium bromide (0.5 µg/mL) for 2–5 min ina staining bath (Caution: wear gloves, because ethidium bromide is carcinogenic).After staining, the gels are briefly washed in ddH 2 O to remove the surplusethidium bromide (again wear gloves and collect ethidium bromide waste separately).The DNA bands can be detected in UV light and can be documented witha suitable system (see also Note 7).3.7 Purification of Nucleic Acids3.7.1. Phenol/Chloroform ExtractionPhenol is also a carcinogen, so work under a fume hood. The extraction ofaqueous nucleic acids with phenol-chloroform allows the denaturing andremoval of proteins (eg., enzymes) from the probes.1. PCR probes (45–40 µL, see also Notes) are diluted with ddH 2 O to a final volumeof 300–500 µL in a microfuge tube.2. The same volume phenol/chloroform/isoamyl alcohol (25:24:1) is applied andprobes are mixed vigorously on a vortex mixer.3. To separate the aqueous—DNA containing—and the nonaqueous phase theprobes are centrifuged at 12,000g for 2 min at room temperature.4. The upper aqueous phase is transferred into a new microfuge tube and the samevolume chloroform/isoamyl alcohol (24:1) is added.5. The probe is mixed and centrifuged again. The upper phase is carried over in anew microfuge tube and the DNA is concentrated with the protocol for ethanolprecipitation.