1.
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After electrophoretic fractionation, visualize stained DNA bands in UV light in transilluminator. Cut out the agar containing the DNA to be recovered with a sterile scalpel.
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2.
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Transfer the agar inside a piece of dialysis tubing pre-treated as described under notes. Add electrophoresis buffer so that the agarose piece is surrounded by buffer with no air bubbles. Use maximum volume of buffer in the dialysis bag.
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3.
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Place the dialysis bag at the bottom of a gel apparatus filled with buffer in such a way that the piece of agar is in the same position with respect to the electrodes as it was in the gel.
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4.
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Apply 100-120 mA for nearly 2h to elute DNA out of the gel.
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5.
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At the end reverse the current for 30 sec in order to mobilize any DNA which may be stuck to the dialysis membrane.
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6.
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Transfer the buffer containing the electroeluted DNA through a cotton-plugged 1mL tip into an Eppendorf tube by centrifuging for 2 min. This process will remove contaminating agarose particles.
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7.
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Extract the supernatant with phenol, then with chloroform: isoamyl alcohol (24 :1) to remove any soluble contaminants.
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8.
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Mix the DNA solution with 3M Tris-HCl (pH 7.5) to give a final concentration of 0.5M. Add two volumes of isopropanol and stand it at -20°C for 30 min.
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9.
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Centrifuge for 10 min at 15,000g. Repeat the precipitation step once more to remove any remaining ethidium bromide.
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10.
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Dry the DNA pellet under vacuum and resuspend in a suitable buffer for any subsequent experiment.
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