Genomic-tip 20/G extraction of high-molecular-weight genomic DNA from ≤ 20 mg animal or plant tissue (with Proteinase K & RNase A treatments)

For more information see the QIAGEN Genomic DNA Handbook.

Safety & PPE

• Wear lab coat, gloves, and eye protection.
• Buffer G2 contains guanidine–HCl; handle inside a fume hood.
• Proteinase K is an allergen—avoid inhalation/skin contact.
• Dispose of waste according to institutional chemical–biological guidelines.

Materials, Reagents & Volumes (per single Genomic-tip 20/G prep)

*Manufacturer-recommended mini-prep volumes,

Additional materials: precooled mortar & pestle (or rotor-stator homogeniser), 10 ml screw-cap tubes, water bath/thermomixer @ 50 °C, microbalance (± 0.1 mg).

Buffer Preparation

1. Prepare Buffer G2 + RNase A: Add 4 µl 100 mg ml⁻¹ RNase A to 2 ml Buffer G2 (final 200 µg ml⁻¹). Stable 6 months at 2–8 °C .
2. Proteinase K stock: Use ready-made 20 mg ml⁻¹ solution or dissolve lyophilised enzyme in distilled water per supplier instructions. Store −20 °C; avoid repeated freeze–thaw.

Sample limits & handling

• Weigh ≤ 20 mg tissue (≤ 15 mg for liver/spleen) to prevent column overload.
• If frozen, keep on dry ice; pellet and discard DMSO/glycerol storage medium before grinding.

Tissue disruption

• Option A (mechanical): Homogenise tissue in 2 ml Buffer G2 (+ RNase A) using a Polytron/Ultra-Turrax (avoid over-shearing).
• Option B (cryogenic): Grind to fine powder in liquid N₂, then add 2 ml Buffer G2 (+ RNase A) and mix. RNase must be present before buffer contacts tissue .

Protein & RNA digestion

1. Transfer homogenate/powder to a 10 ml screw-cap tube.
2. Add 100 µl Proteinase K stock; vortex briefly to mix .
3. Incubate 50 °C for 2 h (extend until lysate is clear) .
4. If particulate matter remains, centrifuge 10 min, 5000 × g, 4 °C; transfer supernatant only.

Equilibrate the column

• Let Buffer QBT run through the Genomic-tip by gravity: 1 ml (20/G), then allow the frit to drip dry.

Load the cleared lysate

• Vortex the lysate 10 s at max speed to reduce viscosity.
• Apply it to the equilibrated tip and let it enter the resin by gravity.
• If flow slows because the sample is very concentrated, you may gently add positive pressure (do not exceed: 4-10 drops min-1).

Wash away contaminants

• Wash with Buffer QC: 3 × 1 ml.

Elute genomic DNA

• Elute with Buffer QF (2 × 1 ml) into a clean polypropylene tube.
• Pre-warming Buffer QF to 50 °C can improve yield.

Precipitate the DNA (0.7 vol isopropanol)

• Add 1.4 ml room-temperature isopropanol.
• Option A: Invert 10–20 × and spool visible threads onto a glass rod.
• Option B: Mix and centrifuge >5 000 ×g, 15 min, 4 °C; decant supernatant.

Recover and resuspend

• A (threads): Transfer directly into 0.1–2 ml TE (pH 8.0) or 10 mM Tris-Cl (pH 8.5); dissolve overnight at RT or 55 °C for 1–2 h.; OR
• B (pellet):

1. Wash pellet with 1 ml cold 70 % EtOH, vortex briefly.
2. Centrifuge >5 000 ×g, 10 min, 4 °C.
3. Air-dry 5–10 min; resuspend as above.

Quality Control

• Measure A₂₆₀/A₂₈₀; expect 1.8 ± 0.1.
• Run 5 µl on 0.5 % agarose pulsed-field mini-gel; intact HMW band indicates minimal shearing.

Storage of DNA

Dissolve pellet in 200 µl TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). Store 2–8 °C (≤ 1 week) or −20 °C (long-term).