Aging Aging

Differentially Expressed Genes 105Table 5Silver Staining Protocol of a Whole 15% 36-Slot CleanGelSilver reaction 20–30 min 200 mL 0.1% AgNO 3 + 200 µLFormaldehyde (37%)Washing Thoroughly wash gel surface with ca. 250 mL of ddH 2 Owith a squeeze bottleDeveloping 2–5 min 200 mL 2.5% Na 2 CO 3 + 200 µL of(visual control) Formaldehyde (37%) +200 µL of Na thiosulfate (2%)Stop/desilver 10 min 250 mL 2.0% (g/v) glycineImpregnate 10 min 250 mL 10% GlycerolTable 6PCR Conditions for the Reamplification of Differentially ExpressedOligonucleotidesPhase Temperature (°C) Time (min) No. of cyclesDenature 94 5 —Denature 94 1Anneal 55 2 35Extent 72 1Cool 4 ∞ —3.5. Reamplification of the differentiallyexpressed oligonucleotides1. The differentially expressed bands can be cut out of the acrylamide gel with ascalpel and then reduced to small pieces with a pipet tip.2. From 50 to 100 µL of sterile ddH 2 O is added and the probes then incubated for 30 minat 65°C and overnight at room temperature to elute the DNA out of the gel.3. For the reamplification reaction, 10 µL of a 1:10 dilution of the eluted oligonucleotidesare used. The PCR mixture is the same as in the amplification reaction.The conditions are shown in Table 6.4. The PCR probes can be analyzed in a regular horizontal DNA electrophoresiswith 1.5–2% agarose gels and then detected with an DNA intercalating stain (e.g.,ethidium bromide). They also can be stored at –20°C.The reamplification reaction could amplify multimers of the original oligonucleotideor other bands, which are artefacts from the elution of the acrylamidegel. For further cloning the fragments into a plasmid, only the interesting bandshould be eluted from an agarose gel by a convenient method. The cloned fragmentsshould further be sequenced, because one eluted band from the random