G NOME Whole Blood Kit
Genomic DNA Isolation from Whole Blood
Revision No. 2011-999-6G01W

The kit is shipped at ambient temperature. However, upon arrival, store Protease Mixx at 4°C. A few days at ambient temperature does not affect activity of this reagent. A precipitate (often appears fungus-like) is sometimes evident after storage. Remove through centrifugation. Protease activity is not affected.

The G NOME Whole Blood DNA Isolation Kit is used to efficiently isolate high molecular weight genomic DNA from whole blood. Each preparation yields up to 100 µg of genomic DNA from 2 ml of whole blood; preparation sizes can be scaled up or down. The DNA is suitable for restriction enzyme digestion or PCR amplification usually 2-3 hours after cell lysis; much of the time involves a protease incubation. The procedure does not use organic extractions.

The G NOME Whole Blood DNA Isolation Kit is available in 3 sizes to suit your needs: 10, 25, and 100 preparations/kit. The kit contains Ace Blood Solution to eliminate hemoglobin/heme/porphyrins (that can inhibit PCR reactons) by lysing cell membranes. Purified nuclei are lysed with Cell Lysis/Denaturing Solution. The kit utilizes RNase Mixx to eliminate RNA immediately after lysis, and Protease Mixx to degrade cellular proteins. This is followed by a proprietary "salting-out" technique which precludes the need for phenol, chloroform, or other organic extractions. The DNA is precipitated with ethanol, pelleted by centrifugation, and dissolved in TE.

Expected yield: 25-50 µg DNA/ml whole blood depending on nucleated cell count.

1. Centrifuge the whole blood for 5 minutes at 500 x g to sediment the cells. Carefully aspirate most of the plasma, being careful not to disturb cell pellet.
2. Add 2.5x volumes of Ace Blood Solution and mix gently by inverting the tube. Centrifuge 15 minutes at 1000-1500 x g. Decant the supernate. Repeat wash with similar volume (2.5x) of the Ace Blood Solution. The result should be a slightly pink to white nuclear pellet.
3. Resuspend nuclei in 1.85 ml Cell Suspension Solution. (Use a 15 ml clear plastic tube for efficient mixing). Mix until the suspension appears homogeneous.
4. Add 50 µl of RNase Mixx; mix thoroughly.
5. Add 100 µl of Cell Lysis/Denaturing Solution**; mix well.
   **If a precipitate forms in stock solution, heat to 60°C to dissolve before using.
6. Incubate at 55°C for 15 minutes.
7. Add 25 µl Protease Mixx; mix thoroughly.
8. Incubate at 55°C for 30 to 120 minutes. (The longer time will result in minimal protein carry-over and will also allow for substantial reduction in protease activity).
9. Add 500 µl "Salt-Out" Mixture; mix gently yet thoroughly. Divide sample into two 1.5 ml tubes. Refrigerate at 4°C for 10-20 minutes to produce a visible precipitate.
10. Centrifuge for 10 minutes at high speed (12,000 x g) in a microcentrifuge. Carefully collect the supernate, avoiding the pellet. If a precipitate remains in the supernate, centrifuge again until it is clear. Transfer the supernate to a 15 ml (or larger) translucent polypropylene tube (e.g., Falcon 2059).
11. To this supernate, add 2 ml TE buffer and 8 ml of 100% ethanol. Gently mix the solution by inverting the tube and hold it at room temperature for 5-10 minutes. Centrifuge for 15 minutes at 1000-1500 x g. Carefully decant or aspirate the supernatant and eliminate the excess ethanol by air drying the DNA pellet.
12. Dissolve the genomic DNA in sterile (autoclaved) TE (10 mM Tris pH 7.5, 1 mM EDTA).