Aging Aging

104 Engel, Adibzadeh, and PawelecTable 4Running Conditions for a Whole 15% CleanGel10 min 200 V 20 mA 10 W60–120 min 375 V 30 mA 20 W20–60 min 450 V 30 mA 20 W3.4.3 Application of the Gel and the Electrode Wicks1. Switch on the thermostatic circulator of the Multiphor II chamber or of a comparableelectrophoresis system; adjust to 21°C.2. Lay two electrode wicks into the compartments of the paper pool, or any comparablechamber.3. Apply 20 mL of the electrode buffer to each wick (for the anode wick use theanode buffer and for the cathode wick the cathode buffer).4. Apply a very thin layer of kerosine onto the cooling plate with a tissue paper, toensure good contact with the gel.5. Place the gel (surface up) on the center of the cooling plate: the side containingthe wells must be oriented toward the cathode.6. Place the cathodal strip onto the cathodal edge of the gel, and the anodal one ontothe other edge. For the DELECT buffer system the strips should be ca. 8 mmaway from the edges of the samples. Smooth out any air bubbles.3.4.4. Sample Application and Gel Electrophoresis1. Apply 10–15 µL of each sample to the sample wells. To determine the size of theamplifed products it would be worthwhile in addition to apply a molecular weightmarker.2. Clean palladium electrode wires with a wet tissue paper before each electrophoresisrun.3. Move electrodes so that they will rest on the outer edges of the electrode wicks.The running conditions for a whole 36-slot 15% gel are shown in Table 4. If gelsare divided the voltage should be kept constant and the mA and W should beadjusted according to the gel size (e.g., the half for half-gels).Depending on the fragment size, the second step has to be varied in length.The larger the amplified oligonucleotides are, the longer this step has to run. Thexylene cyanol runs at a molecular range of ca. 100 basepairs.4. After running, gels have to be fixed in 10% acetic acid or 15% EtOH/5% aceticacid for at least 30 min. The fixation step can be prolonged overnight.3.4.5. Silver Staining1. After fixation, gels are washed 3 × 5 min in ddH 2 O. Then the silver stainingprotocol shown in Table 5 is performed (see also Note 4).Gels can be stored at room temperature after they are shrink-wrapped into astrong cling film.