Genomic DNA Isolation Reagent

Genomic DNA Isolation Reagent (Reagent Based) provides an easy 3-step method to isolate high yields of total DNA (from tissue, cultured animal and bacterial cells, blood, and serum). This unique reagent ensures total DNA with a high yield and good quality from samples of unlimited size. If a large sample is required, the reagent volume can be scaled proportionately, making this reagent not only very user-friendly but also highly versatile. The DNA phenol extraction is not required and the entire procedure can be completed in 90 minutes. The extracted total DNA is ready for use in PCR, Real-time PCR, Southern Blotting, Mapping, and RFLP.

• Fast procedure delivering high-quality genomic DNA.
• Ready-to-use DNA for high performance in any downstream application.
• Consistent DNA yields from small amounts of the starting material.
• Provide sufficient reagents and 3 steps to treat samples from 300 μl to 1.5 ml.
• Time flexibility.
• Ease of DNA extraction technique or method.
• Sample: Tissue, cultured animal and bacterial cells, blood and serum.

• Quantity of DNA needed
• Molecular weight and size of DNA
• Purity of DNA required
• Downstream applications of DNA

Sample Preparation

• Tissue ↘

• Cultured Animal/Bacterial Cells ↘

• Fresh Blood/Frozen Blood ↘

Step 1 Lysis

• Tissue

Add 350 μl of GD Reagent and 0.5 μl of RNase A (50 mg/ml) to the sample in the homogenizer and grind the sample until it is completely dissolved.

Transfer the dissolved sample to a 1.5 ml microcentrifuge tube.

• Cultured Animal and Bacterial Cells/Fresh blood/Frozen Blood

Add 350 μl of GD Reagent and 0.5 μl of RNase A (50 mg/ml) to the sample and mix.

• Serum

1. Transfer 100 μl of the serum to a 1.5 ml microcentrifuge tube.
2. Add 350 μl of the GD Reagent and 0.5 μl of the RNase A (50 mg/ml) and mix completely.
3. Incubate Tissue/Cultured Animal and Bacterial Cells/Fresh Blood/Serum samples at 60ºC for 10 minutes. When using Frozen Blood samples, incubate at 90°C for 30 minutes.
4. Incubate at 15-30°C for 5 minutes. For Frozen Blood or Tissue (for all other samples proceed directly to Step 2), centrifuge at 14-16,000 x g at 2-8°C for 15 minutes and transfer the supernatant to a new 1.5 ml microcentrifuge tube.

Step 2 Phase Separation

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

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

Step 3 DNA Precipitation

1. Add 800 μl of isopropanol to the 1.5 ml microcentrifuge tube containing the clear upper phase from the Step 2.
2. Mix the sample by inverting gently and letting it stand for 5 minutes at the room temperature (The 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.
6. Completely discard the supernatant and re-suspend the pellets in 50-100 μl of TE buffer (not provided) or ddH2O.
7. Incubate for 10 minutes at 60°C to dissolve the pellet.

• Check buffers before use for salt precipitation. Re-dissolve any precipitate by warming up to 37°C.
• During the 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.