DNA Cleanup using illustra™ GFX Kits

Extracted from Nucleic Acid Sample Preparation for Downstream Analyses, GE Healthcare, 2007

GE Healthcare provides three silica-based kits for DNA cleanup. illustra™ GFX™ PCR and Gel Band Purification Kit purifies PCR products and other DNAs from solution or gel slices. This product is discussed in more detail below. illustra™ GFX 96 PCR Purification Kit purifies PCR products from solution in a 96-well format, allowing for increased throughput. See the Sample Prep Guide for details on the GFX 96 PCR Kit. illustra™ CyScribe™ GFX Purification Kit is designed for cleanup of Cy3- or Cy5- dye-labeled cDNA before microarray or other applications.

illustra™ GFX PCR DNA and Gel Band Purification Kit is designed for the purification and concentration of DNA from PCR mixtures, restriction enzyme digests, solutions, and agarose gel bands. DNA ranging in size from 50 bp up to 10 kbp can be purified from solution volumes of up to 100 μL and from gel slices of up to 900 mg. No modifications are required for purification of DNA from gels run in borate-based buffers (e.g., TBE).

The developed method uses a chaotropic agent to extract DNA from solution and/or to dissolve agarose and to denature proteins
(see the Sample Prep Guide). DNA binds selectively to the silica membrane contained in an illustra™ GFX MicroSpin column. The matrix-bound DNA is washed with an ethanolic buffer to remove salts and other contaminants, and the purified DNA is eluted in a low-ionic-strength buffer.

Table 8.3 Product specifications for illustra™ GFX PCR DNA and Gel Band Purification Kit.

Sample type	PCR mixtures, enzymatic reactions, DNA solutions, agarose gel slices
Sample size range	50 bp–10 kbp
Input volume	100 μL solution or up to 900 mg agarose
Elution volume	10–50 μL
Major subsequent applications	Further PCR amplification, sequencing, labeling, restriction enzyme digestion, ligation, cloning
Yield obtained when purifying a 910 bp fragment at a starting concentration of 8.4 ng/μL	PCR, 10 μL elution volume: 65% PCR, 50 μL elution volume: 82% 300 mg agarose, 10 μL elution volume: 57% 300 mg agarose, 50 μL elution volume: 91%

When purifing a PCR fragment less than 50 bp (but greater than 10 bp), use an illustra™ MicroSpin G-25 Column.

This kit cannot be used for the purification of RNA.

If handling large numbers of samples in solution, 100 bp–10 kbp in length, use illustra™ GFX 96 PCR Purification Kit. Chapter 9.

Procedure for purifying and concentrating DNA using illustra™ GFX PCR DNA and Gel Band Purification Kit

Materials

• Buffers and illustra™ GFX MicroSpin Columns are included with the product
• Heating block or water bath set at 60 °C (for agarose gel slices only)

Advance preparation

Before 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.

General protocol

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

1. Prepare sample for binding

a. From solution: Add Capture Buffer to prepare the sample for binding. Capture Buffer contains chaotropic salts that denature proteins (including enzymes) and promote adsorption of DNA to the silica matrix.

If the sample contains DNA greater than 5 kb, do not vortex, as this will cause shearing of the DNA.

b. From agarose: Add Capture Buffer to dissolve agarose and prepare the sample for binding. Incubate/mix at 60 °C until agarose dissolves.

2. Bind DNA

Add sample from step 1 to the GFX column. Centrifuge. DNA greater than 50 bp binds to the silica membrane in the presence of chaotropic salts.

3. Wash and dry

Wash the matrix-bound DNA with an ethanolic buffer to remove salts and other contaminants.

Note: the membrane should be free of liquid following the centrifugation step to ensure that ethanol is not eluted into the purified DNA.

Wash and dry steps can 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

Elute DNA from the membrane using 10 to 50 μL of a low-ionic-strength elution buffer. Incubate briefly at room temperature and centrifuge to elute purified DNA. In the absence of chaotropic salts, DNA desorbs from the membrane.