Plant Lab Protocols / Methodology for Nucleic Acids
Recovery of DNA Fragments from Agarose Gels
Several methods have been developed in recent years for the recovery of DNA fragments from agarose and polyacrylamide gels following electrophoresis. However, no single method has been universally adopted. The following is one of the many methods for the recovery of DNA by electroelution that could work in the subsequent gene manipulation experiments.
Materials
» TBE Electrophoresis Buffer
» 3M Tris-HCl (pH 7.5)
» Dialysis Tubing
Procedure
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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Notes
1. Sterilize dialysis tubing for 30 min at 120°C in a solution of 5% w/v sodium carbonate, 1mM EDTA and keep at 4°C in sterile distilled water.
2. DNA fractionation can be done on low-melting agarose gels and processed subsequently for ligation reaction.
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