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        MicroElute Gel Extraction Kit

        互联网

        929

        实验试剂

         

        1. Sterile deionized water (or TE buffer)

        2. Absolute (or 95%) ethanol

        实验设备

         

        1. Protective eye-ware

        实验步骤

         

        1. MicroElute™ Gel Extraction Spin Protocol

           1) Perform agarose gel/ethidium bromide 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 used 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 UV light box ensuring that as much agarose gel as possible has been removed. Avoid more than 30 seconds exposure of UV light to the DNA. Always use protective eye-ware when working with UV light.

           3) Determine the approximate volume of the gel slice by weighing it in a clean 1.5 mL microcentrifuge tube. Assuming a density of 1 g/mL of gel, the volume of gel is derived as follows: a gel slice of mass 0.2 g will have a volume of 0.2 mL. Add equal volume of Binding Buffer. Incubate the mixture at 55oC-60oC for 7 min or until the gel has completely melted. Mix by shaking or vortexing the tube in every 2-3 minutes.

        Important: Monitor the pH of the Gel/Binding buffer mixture after the gel completely dissolved. DNA yield will significantly decreased when pH > 8.0. If the color of the mixture become orange or red, Add 5 ul 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) Apply 700 ul of the DNA/agarose solution to a HiBind® MicroEluteTM DNA column assembled in a clean 2 mL collection tube (provided) and centrifuge at 8000-10,000 x g for 1 min at room temperature. Discard the liquid and re-use the collection tube. For volumes greater than 700 ul load the column and centrifuge successively, 700 ul at a time. Each HiBind® extraction column has a total capacity of 10-15 ug DNA. If the expected yield is larger, divide the sample into the appropriate number of columns.

           5) Add 300 ul of Binding Buffer (XP2) into the column. Centrifuge at 10,000 x g for 1 min at room temperature to wash the column. Discard the flow-through and reuse the collection tube.

           6) Add with 700 ul SPW Wash Buffer diluted with absolute ethanol into the column and centrifuge at .10,000 x g for 1 minute at room temperature. Repeat this step with another 700 ul SPW Wash Buffer.

        Note: SPW Wash Buffer Concentrate must be diluted with absolute ethanol before use. Refer to label on bottle for directions.

           7) Discard liquid and centrifuge the empty column for 1 min at maximal speed ($13,000 x g) to dry the column matrix. This is critical for good DNA yields.

        2. MicroEluteTM Gel Extraction Vacuum/Spin Protocol

        Note: Please read through previous section before using this protocol.

           1) Prepare the gel sample and dissolve the gel by following the Gel Extraction Spin Protocol.

           2) Prepare the vacuum manifold according to manufacturer’ s instruction and connect the V-Spin column to the manifold.

           3) Load the dissolved DNA/agarose solution from step 3 to the column.

           4) Switch on vacuum source to draw the sample through the column and turn off the vacuum.

           5) Wash the column by adding 300 ul Binding Buffer, draw the Binding Buffer through the column by turn on the vacuum source.

           6) Wash the column by adding 700 ul SPW Wash Buffer, draw the SPW Wash buffer through the column by turn on the vacuum source.

           7) Assemble the column into a 2 mL collection tube and transfer the column to a micro centrifuge. Spin at maximal speed ($13,000 x g) for 1 minute to dry the column.

           8) Place the column in a clean 1.5 mL microcentrifuge tube and add 10-20ul Elution Buffer (10mM Tris, pH8.5 ). Stand for 1-2 minute and centrifuge at maximal speed ($13,000 x g) for 1 minute to elute DNA.

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