FastDNA^® SPIN Kit for Soil
Protocol
Revision No. 6560-999-5F03W

The FastDNA SPIN Kit for Soil is designed to extract PCR-ready genomic DNA from soil samples in less than 30 minutes. The rapid DNA extraction method precludes the use of harmful organic solvents such as phenol and chloroform. The kit enables the extraction of genomic DNA from all bacteria, fungi, plants, and animals in a soil community (see ref. 1. for a report on the use of the FastDNA SPIN Kit for Soil for this purpose). The resulting DNA ranges from ~6 to 25 kb.

The kit consists of three general components:

1. Lysing Matrix

   The MULTIMIX 2 Tissue Matrix Tubes contain a mixture of ceramic and silica particles designed to efficiently lyse all microorganisms including historically difficult sources such as eubacterial spores and endospores, gram-positive bacteria, yeast, algae, nematodes, and fungi.

2. Homogenization Reagents

   The MT Buffer and Sodium Phosphate Buffer have been carefully selected and prepared to enable complete sample homogenization and protein solubilization. The reagents enable extraction of genomic DNA with minimal RNA contamination.

3. DNA Purification and Elution Reagents

   A GENECLEAN procedure is then used to purify the genomic DNA. The procedure purifies DNA with a proprietary silica matrix and eliminates contaminants that inhibit subsequent reactions.

   • Binding Matrix Suspension
   • SEWS-M (Salt Ethanol Wash)
   • DES (DNA Elution Solution; ultra-pure water)

Sample Processing:

1. Add up to 500 mg of soil to MULTIMIX 2 Tissue Matrix Tube.

Lysis:

Add 978 µ Sodium Phosphate Buffer and 122 µl MT Buffer. [See Note 1].

1. Secure tubes in FastPrep Instrument and process for 30 seconds at speed 5.5.
2. Centrifuge MULTIMIX 2 Tubes at 14,000 x g for 30 seconds. [See Note 2].
3. Transfer supernatant to a clean microcentrifuge tube. Add 250 µl PPS reagent and mix by shaking the tube by hand 10 times.
4. Centrifuge at 14,000 x g for 5 minutes to pellet precipitate. Transfer supernatant to a clean microcentrifuge tube. (Resuspend Binding Matrix Suspension before use.) Add 1 ml Binding Matrix Suspension to the supernatant.
5. Place on a rotator or invert by hand for 2 minutes to allow binding of DNA to matrix. Place tube in a rack for 3 minutes to allow settling of silica matrix.
6. Remove 500 µl of supernatant, being careful to avoid settled Binding Matrix. Discard supernatant. Resuspend the Binding Matrix in the remaining amount of supernatant. Transfer the mixture to a SPIN Filter and centrifuge at 14,000 x g for 1 minute. Decant flow-through in catch tube as needed.
7. Add 500 µl SEWS-M to the SPIN Filter and centrifuge at 14,000 x g for 1 minute. Decant flow-through from Catch tube and replace SPIN Filter. Centrifuge at 14,000 x g for 2 minutes to "dry" the matrix of residual SEWS-M wash solution.
8. Remove the SPIN Filter and place in a new kit-supplied Catch Tube. Air dry the SPIN Filter with the cap open for 5 minutes at room temperature.
9. Add 50 µl DES (DNase/Pyrogen Free Water) and resuspend the silica pellet by brief vortexing or finger-flicking the spin filter. Centrifuge at 14,000 x g for 1 minute to transfer eluted DNA to Catch Tube. DNA is now application-ready.

Note 1

Important: The volumes are calculated to leave an air space of approximately 0.25 cc. If less air space is present, there is a likelihood of sample loss due to tube failure or deformation around the cap allowing sample to bubble out. Sample loss is caused by an increase in pressure due to temperature rise during FastPrep runs. The presence of 0.25 cc of air space in the tube is sufficient to prevent sample loss during routine FastPrep runs.

Note 2

Extending spin to 15 minutes can enhance elimination of excessive debris from large samples or from cells with complex cell walls.

Note: Please check that tubes are balanced by weight and that the bottom or side of the tubes will not scrape the wall of your microcentrifuge as this will cause rapid loss of sample.

The FastPrep Instrument shakes a tube up and down at very high speeds. The rotor holds 12 x 2 ml tubes enabling 12 samples to be processed simultaneously.

During processing, the 2 ml tubes that contain the Lysing Matrix sample also contain a chaotropic DNA stabilizing solution which is a proprietary mixture of detergents and salts. The detergents have been found to serve two functions. One, they contribute to inactivate nucleases. Two, they provide lubrication during the lysing step to control the degree of shearing of the DNA.

The FastPrep System, which includes both the FastPrep Instrument and FastDNA and FastRNA kits, has the ability to lyse cells with minimal shearing of the nucleic acids. The procedure eliminates the major concerns in isolation of nucleic acids from cells that are difficult to lyse without enzymes, manual grinding, or homogenizing. It is these laborious and time consuming lysing steps which allow nucleases to act and can make nucleic acid isolation a chore. The FastPrep System, by use of highly energetic mechanical means and careful choice of reagents, disrupts whole tissues, lyses cells, and stabilizes nucleic acid from any source, thus eliminating the need for lysing enzymes or grinding and homogenizing equipment.

1. Cheung, A.L., Eberhardt, K.J. and Fischetti, V.A., Analytical Biochemistry 222,511-514, 1994.
2. Dana, R. C., The Genetic Revolution, San Diego Conference, AACC, November,1994.
3. Tantod, B., Barnes, L, Dana, R.C., ASCB Meeting, San Francisco, December, 1994.
4. Dana, R.C., Saghbini, M., Lippman, D., and Gautsch, J., Plant Genome III, San Diego, January, 1995.
5. Dana, R.C., Saghbini, M., Lippman, D., and Cheung, A. L., J. NIH Research, 7:61, 1995.
6. Borneman, J., Skroch, P.W., O'Sullivan, K.M., Palus, J.A., Rumjanek, N.G., Jansen, J.L., Nienhuis, J and Triplett, E.W., Applied And Environmental Microbiology, June 1996, p.1935-1943