1.Perform agarose gel/ethidium bomide electrophoresis to fractionate DNA fragments. Any type or grade of agarose may be used. It is strongly recommended however, that fresh TAE buffer, or TBE buffer be usd as running buffer, Do not reuse running buffer as its pH will increase and reduce yields.
2.When adequate separation of bands has occurred, CAREFULLY excise the DNA fragment of interest using a wide, clean, sharp scalpel. Minimize the size of the gel slice by removing extra agarose.
3.Determine the appropriate volume of the gel slice by weighing it in a clean 1.5 ml microcentrifuge tube. Assuming a density of 1g/ml of gel, the colume of gel is derives as follows: a gel slice of mass 0.3g will have a volume of 0.3ml. Add an equal volume of Binding Buffer (XP2). Incubate the mixture at 50℃-55℃ for 7min or until the gel has completely melted. Mix by shaking or vortexing the tube in increments of 2-3 minutes.
IMPORTANT: Monitor the pH of the Gel/Binding Buffer mixture after the gel has completely dissolved. DNA yields will significantly decrease when pH>8.0. If the color of the mixture becomes orange or red, add 5 μl of 5M sodium acetate, pH 5.2 to bring the pH down. After this adjustment, the color of the Gel/Binding Buffer mixture should be light yellow.
4.Place a HiBind DNA column in a provided 2 ml collection tube.
5.Apply 700 μl of the DNA/agarose solution to the HiBind DNA column, and centrifuge at 10000 x g for 1 min at room temperature.
6.Discard liquid and place the HiBind DNA column back into the same collection tube. For volumes greater than 700 μl, load the column and centrifuge successively, 700 μl at a time. Each HiBind DNA column has a total capacity of 25 μg DNA. If the expected yield is larger, divide the sample into the appropriate number of columns.
7.Add 300 μl of Binding Buffer (XP2) into the HiBind DNA column, Centrifuge at 10000 x g for 1 min at room temperature to wash the column. Discard the flow-through and re-use the collection tube.
8.Wash the HiBind DNA column by adding 700 μl of SPW Wash Buffer diluted with absolute ethanol. Centrifuge at 10000 x g for 1 min at room temperature.
Note: SPW Wash Buffer Concentrate must be diluted with absolute ethanol before use, See label for directions. If refrigerated, SPW Wash Buffer must be brought to room temperature before use.
Optional: repeat step 8 with another 700 μl of SPW Wash Buffer diluted with absolute ethanol.NOTE: Perform the second wash step for any salt sensitive downstream applications.
9.Discard liquid and centrifuge the empty HiBind DNA column for 2 min at maximal speed to dry the column matrix. Do not skip this step, it is critical for the removal of ethanol fro the HiBind DNA column.
10.Place a HiBind DNA column into a clean 1.5 ml microcentrifuge tube. Add 15-30 μl (depending on desired concentration of final product) of Elution Buffer (10 mM Tris-HCl, pH 8.5) directly on to the column matrix and incubate at room temperature for 1 minute. Centrifuge for 1 min at maximal speed to elute DNA. This represents approximately 70% of bound DNA. An optional second elution will yield any residual DNA, though at a lower concentration.
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