DNA Extraction from Blood and Tissue
(Using a Qiagen ® DNeasy Kit)
Printable Version: DNA Extract Protocol
Materials
Blood or tissue sample
Qiagen DNEasy kit for blood and tissue
Proteinase K
Molecular grade ethanol
Equipment/Labware
Heat block
Micro-centrifuge tubes
Pipettes
Vortex
Centrifuge
Tube racks
DNA Extraction from Blood (Takes approximately 1 hour)
Digestion
- Preheat heating block to 56 º C.
- Suggestion: Read through directions and label appropriate tubes prior to starting.
- Pipette 20 μL QIAGEN Protease (Proteinase K can be substituted) into the bottom of a 1.5 mL micro-centrifuge tube.
- Notes: Protease should be kept at -20 º C (freezer), Proteinase K at 4 º C (refrigerator) or room temperature.
- Add 200 μL sample (blood) to the micro-centrifuge tube.
- Add 200 μL Buffer AL to the sample (micro-centrifuge) tube.
- If precipitates form, dissolve at 56 º C)
- Vortex for 15 seconds.
- Incubate in heating block at 56 º C for 10 min.
- Centrifuge sample at 8 (x 1000) rpm for 1 min.
Washing
- Add 200 μL ethanol to the sample tube.
- Vortex for 15 seconds.
- Centrifuge again at 8 (x 1000) rpm for 1 min.
- Carefully pipette entire sample mixture from micro-centrifuge tube into the QIAamp spin column (in a 2 mL collection tube) without wetting the rim, close the cap.
- Label the cap of the spin column with sample ID.
- Centrifuge at 8 (x 1000) rpm for 1 min.
- Note: If there is sample left in filter column, spin again at full speed (13.2 rpm) for 1 min.
- Place the QIAamp spin column in a clean 2 mL collection tube and discard the previous tube containing the filtrate. (Filtrate is the liquid that ran through the column.)
- Add 500 μL Buffer AW1 without wetting the rim and close cap.
- Centrifuge 8 (x 1000) rpm for 1 min.
- Place QIAamp spin column in another clean 2 mL collection tube and discard the previous tube containing filtrate.
- Add 500 μL Buffer AW2 without wetting rim and close cap.
- Centrifuge at f ull speed 13.2 (x 1000) rpm for 3 min.
- Place QIAamp spin column in another clean 2 mL collection tube and discard previous tube containing filtrate.
- Centrifuge again at fullspeed for 1 min.
Collection
- Place QIAamp spin column into a new 1.5 mL micro-centrifuge tube that is labeled with sample ID, date, gDNA and species. Discard previous tube containing filtrate.
- Add 200 μL Buffer AE (or dH2O) to column.
- Let sit at room temp for 2-5 min.
- Centrifuge at 8 (x 1000) rpm for 1 min.
- Discard filter column.
- Keep micro-centrifuge tube which now contains your gDNA.
- Run samples a 1% gel to verify that your DNA extraction was successf μL and you do indeed have gDNA. If bands are visible…You now have Genomic DNA (gDNA) collected in the labeled micro-centrifuge tube. This sample sho μLd be kept frozen.
DNA Extraction from Tissue (Takes approximately 30 minutes to finish after step 3)
Digestion
- Preheat heating block to 55 º C. Read through directions and label appropriate tubes prior to starting.
- Cut up 25mg (you may need to convert this to grams in order to weigh this on your scale) tissue into small pieces (slivers).
- Place in a 1.5 mL micro-centrifuge tube.
- Think about what tissues will digest better for your tissue selection.
- Add 180 μL Buffer ATL.
- Add 20 μL Proteinase K to sample (micro-centrifuge) tube.
- Vortex for 15 seconds.
- Incubate in heating block at 55 º C for at least 1 hour or as long as necessary
- Vortex every hour (if possible) during incubation to dissolve sample.
- It is ok for the sample to be somewhat thickened (oil vs. water) but should not be gelatinous (like Jell-o). You should not have clumps of tissue remaining; tissue should be broken into very, very small particles. Ideally, tissue will be completely dissolved as fragments or gelatinous material will clog the filter.
- Tissue may require anywhere from 1 hour to over 24 hours to digest, especially for skin tissue.
- Vortex for 15 seconds before continuing to next step.
- Set heating block to 70 ºC.
- Add 200 μL Buffer AL to sample tube.
- Buffer AL is light sensitive, keep container covered in aluminum foil.
- Mix thoroughly by vortexing.
- Incubate at 70 º C for 10min. Turn off block heater.
- Washing
- Add 200 μL ethanol to sample.
- Mix thoroughly by vortexing.
- Carefully pipette entire sample into a DNeasy spin column (in a 2 mL collection tube).
- Label the cap of the spin column with sample ID.
- If there is any precipitate in micro-centrifuge tube, make sure to transfer that to the spin column also.Centrifuge at 8 (x 1000) rpm for 1 min. If there is sample left in filter column, spin again at fullspeed (13.2 rpm) for 1 min.
- Place the DNeasy spin column in a new 2 mL collection tube and discard tube containing filtrate.
- Add 500 μL Buffer AW1 to DNeasy spin column.
- Centrifuge at 8 (x 1000) rpm for 1 min.
- Place the DNeasy spin column in a new 2 mL collection tube and discard tube containing filtrate.
- Add 500 μL Buffer AW2 to DNeasy spin column.
- Centrifuge at fullspeed (13.2 x 1000 rpm) for 3 min.
- Place QIAamp spin column in another clean 2 mL collection tube and discard previous tube containing filtrate.
- Centrifuge again at fullspeed for 1 min.
Collection
- Place the DNeasy spin column in a clean 1.5 mL micro-centrifuge tube that is labeled with the sample ID, date, gDNA, species, and #1 (representing the 1st elution). Discard the previous tube containing the filtrate.
- Add 200 μL of Buffer AE (or dH2O) to spin column. (100 μL for more concentrated gDNA)
- Let sit at room temperature for 2-5 min.
- Centrifuge at 8 (x 1000) rpm for 1 min.
- Keep micro-centrifuge tube which now contains your st elution of gDNA.
- Place the DNeasy spin column in another clean 1.5 mL micro-centrifuge tube that is labeled with the sample ID, date, gDNA, species, and #2 (representing the 2nd elution).
- Add 200 μL of Buffer AE (or dH2O) to spin column. (100 μL for more concentrated gDNA)
- Let sit at room temperature for 2-5 min.
- Centrifuge at 8 (x 1000) rpm for 1 min.
- Discard filter column. Keep micro-centrifuge tube which now contains your 2nd elution of gDNA.
- Elution #1 and #2 can be combined for a larger amount of concentrated gDNA (use caution as this can be a source of contamination).
- Run a 1% gel to verify that your DNA extraction was successful and you do indeed have gDNA. If bands are visible …You now have Genomic DNA (gDNA) collected in the labeled micro-centrifuge tubes. Sample elution #1 will have a higher concentration of gDNA than sample elution #2 therefore generally giving better results. These samples should be kept frozen.