DNA Extraction (Qiagen)

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The protocol for extracting DNA with the Qiagen Tissue Kit.

This protocol has proven successful for rockfish (Sebastes), cod (Gadus morhua), and oysters (Crassostrea).

Materials and Equipment

Qiagen Extraction Kits

Qiagen Blood and Tissue Extraction Kit - single sample

  • 250 samples - Cat No./ID: 69506 - ~700$

  • 2.80$ per sample

Qiagen Blood and Tissue Extraction Kit - 96 plate

  • 4x96 (384 samples) - Cat No./ID: 69506 - ~1300$

  • ~3.40$ per sample

Additional Reagents and Consumables

  • Razors (for cutting tissue)

  • Flame (sterilization)

  • Weigh paper

  • Kim wipes

  • Pipet Tips (10uL,20uL,200uL,1000uL)

  • EtoH (100% - molecular grade)

  • Low TE for elutions if working with CviMVP samples

Equipment

  • Micro-scale (need to be able to measure down to 20mg)

  • Thermo-mixer (It has both single sample and plate attachments)

  • Vortexer

  • Microcentrifuge (large centrifuge for plate extractions)

  • Pipets (10uL,20uL,200uL,1000uL)

Protocol

Before Starting

  • If any precipitate has formed in either the Buffer ATL or Buffer AL, warm solution(s) to 56 degrees C until precipitates have fully dissolved.

  • Preheat thermo-mixer to 56 degrees C.

Tissue Lysing

1.  Prep surfaces and area around the scale. First clean with bleach solution, followed by H2O, then 70% EtoH.

2.  If working with many samples (\>10) get a styrofoam container with ice to keep tissue samples cool while prepping for extraction.

3.  Remove tissues from freezer and let thaw (do not let them sit any longer than necessary). Once thawed they can be placed on ice (this generally won't cause them to re-freeze).

4.  Tare piece of weighing paper on scale.

5.  Carefully remove tissue from 2mL tube. Using a sterile razor blade to cut a piece of tissue roughly 20mg in weight. Using 100% EtoH, gently rinse tissue and pat dry with clean kim wipe, so that the tissue appears nearly dry.

6.  Place tissue on weigh paper and measure the weight, if the tissue weighs 20mg+/-2mg place it in extraction tube. If not, either remove tissue as necessary or add more from sample tissue.

Note: Between samples razors & forceps should be sterilized by spraying with EtoH and using a flame to burn off any residual EtoH and tissue.

  1. Record weight in lab notebook.

  2. After tissues have been processed and place into new clean 1.5mL tubes, add 180 uL of Buffer ATL and 20 uL of Proteinase K to each sample tube (same as standard Qiagen kit). Also, turn on Thermomixer and preheat it to 56 degrees. (For MVP project, add the Buffer ATL into tubes before tissue cutting for consistency with plate protocol).

  3. Seal tubes, confirm all tube labels, then vortex for 15 seconds.

  4. Place samples on thermo-mixer set at 56 degrees at 600 rpm. BEFORE STARTING THE MIXER: Make sure all tube caps are securely fastened. In addition, after 2 minutes on the thermo-mixer confirm the tube caps haven’t popped open.

  5. Samples can be left for at least 2 hours or overnight to allow for all tissue to be completely lysed.

Extraction

  1. Prep surfaces and area around the work bench. First clean with bleach solution, followed by H2O, then 70% EtoH.

  2. Pre-label top of spin-column tube and 2 additional 1.5 mL tubes (one label E1 for elution 1 and E2 for elution 2).

  3. Add 200 μl Buffer AL. Mix thoroughly by vortexing.

  4. Add 200 μl ethanol (96–100%). Mix thoroughly by vortexing.

Note: A working solution of Buffer AL & ethanol can be made to be aliquoted into sample tubes.

  1. Pipet the mixture into a DNeasy Mini spin column placed in a 2 ml collection tube. Centrifuge at ≥ 6000 x g (8000 rpm) for 1 min. Discard the flow-through and collection tube.

  2. Place the spin column in a new 2 ml collection tube. Add 500 μl Buffer AW1, and centrifuge for 1 min at ≥6000 x g. Discard the flow-through and collection tube.

  3. Place the spin column in a new 2 ml collection tube. Add 500 μl Buffer AW2, and centrifuge for 3 min at 20,000 x g (14,000 rpm). Discard the flow-through and collection tube.

Note: Spin column membrane should be dry after this step. Spin for an additional 1 min if not.

  1. Transfer the spin column to a new 1.5 ml or 2 ml microcentrifuge tube.
Number of Elutions

The Qiagen protocol suggests performing 2 elutions, but 1 elution is typically enough to elute DNA with concentrations in the hundreds. If performing only one elution (as in Cvi extractions), perform the longer incubation (10 min) at room temperature, which is done for the second elution in the Qiagen protocol.

  1. Elute the DNA by adding 200 μl Buffer AE or (low TE buffer if working with CviMVP samples) to the center of the spin column membrane. Incubate for 1 min at room temperature (15–25°C). Centrifuge for 1 min at ≥6000 x g.

  2. Perform second elution by adding 200 μl Buffer AE to the center of the spin column membrane. Incubate for 10 min at room temperature (15–25°C). Centrifuge for 1 min at ≥6000 x g.

  3. Extracted DNA can be place in fridge for a few days, or store at -20 or -80 for long term storage. AVOID numerous freeze-thaw cycles.