Procedure for Purifying and Concentrating DNA using illustra™ GFX 96 PCR Purification Kit

A general protocol for purification and concentration of DNA from PCR mixtures, restriction enzyme digests, and solutions using illustra™ GFX™ 96 PCR Purification Kit is provided below. The kit can be used with suitable centrifuges, vacuum manifolds, or automated robotic workstations.

Materials

Buffers and GFX™ Binding, Wash, and Collection plates are included with the product

Benchtop microcentrifuge suitable for centrifugation of 96-well plates OR NucleoVac 96 Vacuum Manifold, available from Macherey-Nagel (product code 740681)

Reagent reservoir (optional for multichannel pipetting)

Advance preparation

Prior to first use, add absolute ethanol to the bottle containing wash buffer type 1. See product instructions for volume. Mix by inversion. Indicate on the label that this step has been completed. Store upright and airtight.

Prepare the vacuum source

GFX™ 96 PCR Purification Kit can be used with Macherey-Nagel NucleoVac 96 vacuum manifold. To process fewer than 96 samples, use a rubber pad or self-adhering polyethylene foil to cover up any unused wells of the GFX™ 96 Binding Plate and ensure a vacuum is maintained. Establish a reliable vacuum source for the manifold. A vacuum pump, house vacuum, or water aspirator may be used. We recommend a vacuum of 380 to 630 mbar or 15 to 25 mm Hg. The use of a vacuum regulator is recommended. Alternatively, adjust the vacuum so that during purification the sample flows through the column at a rate of 1 to 2 drops per second. Vacuum times suggested in the product protocol might have to be increased for complete filtration if large sample volumes are used.

General protocol*

Note: Buffers are NOT transferable between Cytiva kits in the illustra™ product range. Please ensure that you use the correct buffers for your purification.

1. Prepare sample for binding
   
   Add capture buffer to prepare the sample for binding.
   
   Sample volume may be 20 to 300 μl.
   
   
2. Bind DNA
   
   Add sample from step 1 to the GFX Binding Plate. Centrifuge or apply vacuum. DNA binds to the silica membrane in the presence of chaotropic salts. Primers and nucleotides are too small to bind effectively. Enzymes are denatured and will not bind to the column.
   
   
3. Wash and dry
   
   Add wash buffer to membrane-bound DNA. Centrifuge or apply vacuum. The wash buffer contains salts and ethanol to remove unbound contaminants and dry the membrane.
   
   Wash and dry steps may be repeated when purity is paramount, for example if the sample is to be used in a blunt-ended ligation.
   
   
4. Elute purified and concentrated DNA
   
   Add low-ionic-strength elution buffer to the membrane. Incubate briefly at room temperature. Centrifuge or apply vacuum. Membrane-bound DNA is eluted from the membrane when chaotropic salts are removed.
   
   When 50 μl volume is used for elution, an average volume of 45 to 48 μl will be recovered.

* This product can be used with suitable centrifuges or vacuum manifolds. Refer to GFX™ 96 PCR Purification Kit product instructions for specific centrifuge and vacuum protocols. For details on using this product with automated workstations, refer to the manufacturer of your system.