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

To obtain 15-20 µg of high-quality, 20–150 kb genomic DNA suitable for downstream molecular biology applications from fresh or frozen tissue using the QIAGEN Genomic-tip 20/G system, incorporating in-tube protein (Proteinase K) and RNA (RNase A) digestion. Applicable to all routine extractions from soft (e.g., liver, muscle) or hard (e.g., brain, tail) tissues in the laboratory.

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Version 1 Current

Effective: 2025-08-04

Procedure Steps (Version 1)

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)

Reagent	Working volume*
Buffer G2	2 ml
Buffer QBT	1 ml
Buffer QC	3 ml
Buffer QF	2 ml
Isopropanol	1.4 ml
70 % ethanol	1 ml
RNase A stock (100 mg ml⁻¹)	4 µl
Proteinase K stock (20 mg ml⁻¹)	100 µl

*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

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 .
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

Transfer homogenate/powder to a 10 ml screw-cap tube.
Add 100 µl Proteinase K stock; vortex briefly to mix .
Incubate 50 °C for 2 h (extend until lysate is clear) .
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):

Wash pellet with 1 ml cold 70 % EtOH, vortex briefly.
Centrifuge >5 000 ×g, 10 min, 4 °C.
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).