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Poly-Gel DNA purification

Make sure all necessary reagents and equipment are ready before starting. Wash Buffer Concentrate must be diluted with absolute ethanol and stored at room temperature.

  1. Transfer the gel fragment onto a nuclease-free microscope slide. With a second glass slide (or nuclease-free razor) mash and pulp the gel completely. Carefully transfer gel pulp to a 1.5 ml nuclease-free microcentrifuge tube. Add 250 μl Gel Elution Buffer. This volume is usually enough to submerge a slice 2 mm x 10 mm x 0.8 mm. For a larger fragment, adjust volume of Gel Elution Buffer until the gel is covered. Any buffer or dH2O can be used. However, it’s recommend to use the Gel Elution Buffer in order to prevent DNA degradation by exogenous nucleases.

  2. Incubate the gel fragment in Gel Elution Buffer for 1-4 h at 65oC. The elution time isdependent on the size of the gel fragment, DNA size and the temperature of the incubation. About 70% of a 100 bp DNA fragment elutes in approximately 4 hrs at 65°C. Larger fragments will take longer to elute.

  3. Centrifuge at 10,000 x g for 5 min at room temperature. Transfer the supernatant to a new 1.5 ml microcentrifuge tube.

  • If isolating labeled DNA probes for use in hybridization assays, no further purification is necessary. An aliquot of the eluted probe can be used directly in the hybridization reaction. An optional phenol: chloroform extraction may be performed. However do not extract with phenol if the DNA probe is labeled with digoxygenin as DNA will separate into the organic phase. Also a standard etahanol precipitation with carrier (glycogen, tRNA, or linear acrylamide) may be performed for further clean-up.

  1. Add 1 volume Buffer GC and 1 volume 96-100% ethanol to the supernatant. Vortex briefly to mix.

  2. Add 750 μL of the mixture to a DNA column. Centrifuge at 10,000 x g for 1 min at room temperature. Discard the flow-through and reuse the collection tube in step 6.

  3. Add the remaining mixture to the column and centrifuge as above. Discard flow-through and place column back into 2 ml collection tube

  4. Add 750 μL DNA Wash Buffer. Centrifuge at 10,000 x g for 1 min at room temperature. Discard flow-through and place column back into 2 ml collection tube Ensure that ethanol is added to the DNA Wash Buffer before use. DNA Wash Buffer should be at room temperature to ensure effective washing.

  5. Centrifuge the empty column, with the lid open,at top speed for 2 min. It is critical to remove trace amount of ethanol from the column for optimal elution.

  6. Transfer column to a sterile 1.5 ml microcentrifuge tube. Add 50 μL Elution Buffer or sterile dH2O directly onto column matrix. Centrifuge at 10,000 x g for 1 min to elute DNA. For DNA <100 bp approximately 80% will be recovered with a single elution. Subsequent elution steps each yield 80% of the remaining bound material. For expected yields greater than 20 μg, repeat the elution with 50 μl water.

  • If the expected DNA from PAGE is shorter than 60 bp, the eluted DNA can be purified according the protocol below,

  1. Add 0.1 volume Buffer C1 and 0.7 volume isopropanol to the supernatant from Step 3. Mix well by vortexing.

  2. Centrifuge at 12,000 x g for 10 min. Carefully remove the supernatant by pipetting. Avoid touching the bottom of the microcentrifuge tube.

  3. Add 1 mL 70% ethanoland centrifuge at 12,000 x g for 5 min. Carefully remove the supernatant by pipetting. Avoid touching the bottom of the vial.

  4. Briefly spin again for 10 seconds and carefully remove residual ethanol by pipetting. Avoid touching the bottom of the vial.

  5. Air dry the sample in a tissue culture hood for 10-20 min.

  6. Resuspend the DNA in ddH20 or TE Buffer. 

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