PowerSoil® DNA isolation

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Do not use bleach to clean the inside of the PowerVac™ Manifold or to rinse the PowerVac™ Mini Spin Filter Adapters when attached to the manifold.

Upstream tips
Do not use bleach to clean the inside of the PowerVac™ Manifold or to rinse the PowerVac™ Mini Spin Filter Adapters when attached to the manifold.

Publication protocol

DNA was extracted from oil (0.6 g) and sludge (0.2 g) samples using a PowerSoil DNA Isolation Kit (PowerSoil DNA Isolation Kit, MO BIO Laboratories INC, Solana Beach, CA, USA). Almost the whole gene (1541 in E. coli) of 16S rRNA, approximately 1,490 bp, was amplified from samples using the broad-specificity primers 8F (Eden et al. 1991) and 1492R (Lane et al. 1985). PCR reactions were performed in thin-walled tubes using a BioRad iCycler (BioRad, Hemel Hempstead, Herts, UK). Takara Ex Taq Polymerase (Millipore U.K LTD, Watford, UK) was used to amplify DNA from the sample extract. The PCR amplification protocol used with the 8F and 1492R primers was: initial denaturation at 94 °C for 4 min, melting at 94 °C for 30 s, annealing at 50 °C for 30 s, elongation at 72 °C for 3 min; 35 cycles, followed by a final extension step at 72 °C for 5 min. Purity of the amplified products was determined by electrophoresis in Tris-acetate-EDTA (TAE) gel. DNA was stained with ethidium bromide and viewed under short wave UV light using a BioRad Geldoc 2000 system (BioRad, Hemel Hempstead, Herts, UK).

The resulting PCR products were purified using an ExoSap protocol, 2 μl of ExoSap mix (0.058 μl Exonuclease I, 0.5 μl Shrimp Alkaline Phosphatase and 1.442 μl QH2O) was added to 5 μl of PCR product and incubated at 37 °C for 30 min followed by 80 °C for 15 min. Purified gene fragments were ligated directly into a cloning vector using a PCR cloning kit (StrataClone, Stockport, UK) containing topoisomerase I-charged vector arms (Agilent Technologies, Wokingham, UK) prior to transformation into E. coli competent cells expressing Cre recombinase (Agilent Technologies, Wokingham, UK). Positive clones (96 per library) were screened by PCR using primers complementary to the flanking regions of the PCR insertion site of the cloning vector, and sequenced using the ABI Prism® BigDye™ Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Life Technologies Corporation, USA). The forward primer, 8F (Eden et al. 1991) was used for the sequencing reaction.

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Manufacturer protocol

Download the product protocol from Mobio for PowerSoil® DNA isolation below.

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