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Standard Protocol For Isolation of DNA From Dried Blood, Body Fluids and Sperm

互联网2013-11-13

582

实验原理

E.Z.N.A.^® Forensic DNA Kit uses the reversible binding properties of the HiBind^® matrix, a new silica-based material, combined with the speed of mini-column spin technology. A specifically formulated buffer system allows genomic DNA up to 50 kb to bind to the matrix. Samples are first lysed under denaturing conditions and then applied to the HiBind^® spin columns to which the DNA binds, while cellular debris, hemoglobin, and other proteins are effectively washed away. High quality DNA is finally eluted in sterile deionized water or low salt buffer. Each HiBind^® column can bind approximately 100 ug DNA. Use of more than 30 mg tissue or 10⁷ cells is not recommended.

实验步骤

Dried blood, body fluids, and sperm samples on filter paper can be processed using the following method. We recommend using OB Specimen Paper (OSP-01 and OSP-02) for spotting blood, as this unique filter paper disintegrates when incubated in aqueous buffers, allowing for the efficient recovery of DNA. This kit can also be used for samples collected by using other specimen collection papers.

1. Cut or punch out the blood spot (or other sample) from the filter paper. (Up to 200 ul of blood can be used for each spot.) Tear or cut filter into small pieces and place into a microfuge tube.

Note: Use 3-4 punched cycles (3mm diameter) for each DNA isolation.

2. Add 200 ul Buffer STL and incubate at 55℃ for 15 minutes. Vortex every 2 min to mix.

3. Add 25 ul OB protease solution and mix by votexing. Incubate for 45 minutes at 60℃ with occasional mixing. Briefly centrifuge to remove any droplets from inside the lid.

4. Add 225 ul Buffer BL and votex to mix. Incubate at 60℃ for 10 minutes. Briefly centrifuge to remove any droplets from inside the lid.

5. Add 225 ul absolute ethanol and mix thoroughly by vortexing. Briefly centrifuge to remove any droplets from inside the lid.

6. Insert each HiBind^® DNA Minicolumn into a 2 ml collection tube (provided). Transfer the entire sample from Step 5 into the column, including any precipitate that may have formed. Centrifuge at 8,000 x g for 1 min to bind DNA. Discard collection tube and flow-through liquid.

7. Place each column into a second 2 ml tube and wash by pipetting 500 ul of Buffer HB into column. Centrifuge at 8,000 x g for 1 min. Dispose of flowthrough liquid and re-use the collection tube.

8. Place each column into a same 2 ml tube from step 7 and wash by pipetting 700 ul of DNA Wash Buffer diluted with ethanol into column. Centrifuge at 8,000 x g for 1 min. Dispose of collection tube and flow-through liquid.

Note: DNA Wash Buffer is provided as a concentrate and must be diluted with absolute ethanol as indicated on the bottle label and Page 3. If refrigerated, the diluted wash buffer must be brought to room temperature before use. Refrigeration is NOT recommended.

9. Using a new collection tube, wash the column a second time with 700 ul of DNA Wash Buffer and centrifuge as above. Discard flow-through and reuse the collection tube.

10. Using the same 2 ml collection tube, centrifuge at maximum speed (>10,000 x g) for 2 minutes to dry the column. This step is critical for removal of residual ethanol that might otherwise interfere with downstream applications.

11. Place the column into a nuclease-free 1.5 ml microfuge tube and add 50-100 ul of Elution Buffer preheated to 70℃. Allow the tube to sit for 3 minutes at room temperature.

12. To elute DNA from the column, centrifuge at 8,000 x g for 1 min. Repeat the elution with a second volume of 50-100 ul Elution Buffer.

Note: Incubation at 70℃ rather than at room temperature will give a modest increase in DNA yield per elution. Alternatively, use of the first eluate for second elution will increase DNA concentration.

Blood spots from finger pricks usually contain no more than 50 ul blood and yield approximately 500 ng to 1 ug DNA. This is sufficient for PCR analysis. To obtain higher DNA concentrations, elute with 50 ul preheated Elution Buffer or TE and repeat with the first eluate.

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