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    MicroElute DNA Clean-Up Kit

    互联网

    1241

    实验试剂

     

    1. Sterile deionized water (or TE buffer)

    2. Absolute (or 95%) ethanol

    实验设备

     

    1. Sterile 1.5 mL centrifuge tubes.

    2. Protective eye-ware

    实验步骤

     

    1. MicroElute™ DNA Clean-up Spin Protocol

    For DNA fragments (30bp-100 bp)

       1) Add 3 x volume of buffer P3, 3 x volume of isopropanol to the enzymatic reaction and mix throughly with pipetting or vortex. The maximum volume of the reaction can be processed per column is 100ul. For example, 100ul enzymatic reaction, add 300ul Buffer P3 and 300ul isopropanol. Proceed step 2.

    Tips: 3 volume of Buffer P3 and 3 volume of isopropanol can be premixed. Then add 6 volume of this mixture into DNA solution.

    For DNA fragments (100bp-10kbp)

       1) Add 3 x volume of buffer P3, 1 x volume of isopropanol to the enzymatic reaction and mix throughly with pipetting or vortex. The maximum volume of the reaction can be processed per column is 100ul. For example, 100ul enzymatic reaction, add 300ul Buffer P3 and 100ul isopropanol. Proceed step 2.

    Tips: 3 volume of Buffer P3 and 1 volume of isopropanol can be premixed. Then add 5 volume of this mixture into DNA solution.

    For Genomic DNA Clean Up (>10 kb)

       1) Add 0.5 x volume of Buffer P3 and 1 x volume of absolute ethanol (room temperature, 96-100% ) to the DNA solution and vortex to mix well. For example, 100 ul of DNA Solution, add 50 ul of Buffer P3 and 100 ul of absolute ethanol. Proceed step 2.

    Tips: 1 volume of Buffer P3 and 2 volume of absolute ethanol can be premixed. Then add 1.5 x volume of this mixture into DNA solution.

    Purify DNA by HiBindTM MicroEluteTM DNA Column

       2) Place a MicroElute HiBind™ DNA column into a 2 mL collection tube (supplied) in a tube rack.

       3) Apply all the sample mixture into the spin column with a micro pipettor.

       4) Spin at 10,000 x g for 1 minute to bind DNA.

       5) Discard the flow-through and reuse the collection tubes for next step.

       6) Add 700 ul of SPW Wash buffer diluted with ethanol to the HiBind™ MicroElute™ DNA column and spin at 10,000 x g for 1 minute. Discard the flow-through and place the column back into same 2mL collection tube.

       7) Repeat Step 6 with another 700 ul of SPW Wash buffer diluted with ethanol.

       8) Centrifuge at maximum speed ($13,000 x g) for 2 minute to dry the column. Discard the 2 mL collection tubes.

       9) Place the MicroElute™ HiBind™ DNA column into a clean 1.5 mL micro centrifuge tube.

       10) Add 15-20 ul Elution Buffer(10mM Tris, pH 8.5) or water to the center of the membrane, incubate at room temperature for 3-5 minute.

       11) Centrifuge at maximal speed ($13,000 x g ) for 1 minute to elute DNA.

       12) For genomic DNA clean up, repeat elution step 10-11 by adding another 15-20 ul Elution Buffer.

    2. MicroElute™ DNA Clean-Up Vacuum/Spin Protocol

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

       1) Prepare the enzymatic reaction and Buffer P3/isopronoal by following the Spin Protocol step 1.

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

       3) Load the dissolved DNA 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 700 ul SPW Wash Buffer, draw the SPW Wash Buffer through the column by turn on the vacuum source.

       6) 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) for1 minute to dry the column.

       7) 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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