Dual Genomic DNA Isolation Kit (Tissue)

The Dual Genomic DNA Isolation Kit (Tissue) combines reagent system and spin column system. The kit is designed specifically for genomic DNA isolation from animal tissue samples. This unique reagent system ensures the total DNA with high yield and good quality from samples. The spin column system is designed to purify or concentrate DNA products which have been previously isolated with the Reagents. The entire procedure can be completed in 1 hour without phenol extraction. Purified DNA is suitable for use in PCR or other enzymatic reactions.

• Sample: 30-100 mg of fresh animal tissue or 25 mg of paraffin-embedded.
• Tissue Format: Reagent and spin column
• Operation time: within 1 hour.
• Elution volume: 50-200 ul.

Kit Contents

• Delivering high-quality genomic DNA with the fast procedure.
• Ready-to-use gnomic DNA for high performance in any downstream application.
• Highly purified and high yield genomic DNA can be extracted from various tissue samples.
• Optimized tissue lysis buffer for the efficient lysis.

Step 1 Sample Preparation

Fresh Tissue

1. Cut off 100 mg of fresh animal tissue and grind the sample under liquid nitrogen to a fine powder using a mortar and pestle.
2. Proceed with the Step 2 Lysis.

Paraffin-embedded tissue

1. Slice small sections (up to 25 mg) from blocks of paraffin-embedded tissue and transfer to a 1.5 ml microcentrifuge tube.
2. Add 1 ml of xylene to the tube.
3. Vortex vigorously and incubate at room temperature for approximately 10 minutes.
4. Vortex occasionally during incubation.
5. Centrifuge at 14-16,000 x g for 3 minutes. Remove the supernatant.
6. Add 1 ml of absolute ethanol to wash the sample pellet and mix by inverting.
7. Centrifuge at 14-16,000 x g for 3 minutes. Remove the supernatant.
8. Add 1 ml of absolute ethanol to wash the sample pellet again and mix by inverting.
9. Centrifuge at 14-16,000 x g for 3 minutes. Remove the supernatant.
10. Open the tube and Incubate at 37°C for 15 minutes to evaporate any ethanol residue.
11. Proceed with the Step 2 Lysis.

Step 2 Lysis

1. Add 1 ml of Buffer DG and 0.5 μl of RNase A (50 mg/ml) to the sample from Step 1 and grind the sample until it is completely dissolved.
2. Transfer the dissolved sample to a 1.5 ml microcentrifuge tube.
3. Incubate at 75°C for 30 minutes (invert the tube every 10 minutes).
4. Centrifuge at 14-16,000 x g for 5 minutes.
5. Transfer carefully the clear supernatant to a new 1.5 ml microcentrifuge tube.

Step 3 Phase Separation

1. Add 600 μl of chloroform to the supernatant from Step 2.
2. Shake vigorously and then centrifuge at 14-16,000 x g for 10 minutes.
3. Carefully remove the upper phase and transfer it to a new 1.5 ml microcentrifuge tube.
4. Repeat the Phase Separation Step until the interphase becomes clear then transfer the clear upper phase to a new 1.5 ml microcentrifuge tube.

*The number of repetitions is dependent on sample type; e.g. dense tissue samples may require a higher number of repeats.

Step 4 DNA Precipitation

1. Add 800 μl of isopropanol to the 1.5 ml microcentrifuge tube containing the clear upper phase from step 3.
2. Mix the sample by inverting gently and let stand for 5 minutes at room temperature (DNA precipitation can be increased with extended standing time).
3. Centrifuge at 14-16,000 x g for 15 minutes.
4. Discard the supernatant and wash the pellet with 1 ml of 70% EtOH.
5. Centrifuge at 14-16,000 x g for 5 minutes. Completely discard the supernatant and re-suspend the pellets in 200 μl of TE buffer or ddH₂O.
6. Incubate for 10 minutes at 75°C to dissolve the pellet.

• Buffers B and W1 contain irritants. Wear gloves when handling these buffers.
• Add 60 ml of the ethanol to the Buffer W2 before use.
• During operation, always wear a lab coat, disposable gloves, and protective equipment.
• Research Use Only. Not intended for any animal or human therapeutic or diagnostic uses.