Reagents and Equipment Used in the Experiment
|
Cat. No. |
Product Name |
|
18701ES |
|
|
10406ES |
Pre-Experiment Preparation
1. Prepare the following reagents yourself: ethanol, isopropanol, 1× PBS, and RNase A.
2. Preheat the required water bath to 70°C before starting the experiment.
3. Before first use, add the specified volume of absolute ethanol to Protein Removal Buffer PL and Wash Buffer WB, mix thoroughly. After adding ethanol, please check the box on the bottle label to indicate that ethanol has been added, to avoid repeated additions.
Protocol
1. Cut fresh tissue into small pieces with a scalpel or grind it into fine powder in liquid nitrogen. Transfer the tissue into a 1.5 mL microcentrifuge tube containing 180 μL Tissue Lysis Buffer LB. Mix thoroughly by pipetting up and down using a wide-bore tip.
2. Add 20 μL Proteinase K Solution, vortex immediately to ensure complete mixing.
3. Incubate the lysate at 55°C for 30 min until tissue digestion is complete. Gently invert the tube several times during incubation to aid lysis.
4. Optional step: If significant RNA contamination is expected, add 5 μL of 100 mg/mL RNase A (10406ES) after step 3, mix by vortexing, and incubate at room temperature for 5–10 min.
5. Add 200 μL Binding Buffer BD, vortex immediately to mix thoroughly, then incubate at 70°C for 10 min.
6. After cooling to room temperature, add 100 μL isopropanol, vortex immediately to mix. A flocculent precipitate may appear.
7. Transfer the mixture to an Adsorption Column AC, centrifuge at 13,000 rpm for 1 min, and discard the flow-through.
8. Add 500 μL Protein Removal Buffer PL (confirm ethanol has been added), centrifuge at 13,000 rpm for 30 sec, and discard the flow-through.
9. Add 600 μL Wash Buffer WB (confirm ethanol has been added), centrifuge at 13,000 rpm for 30 sec, and discard the flow-through.
10. Repeat step 8 once.
11. Place the Adsorption Column AC back into an empty collection tube and centrifuge at 13,000 rpm for 2 min to completely remove residual wash buffer. This prevents ethanol carryover, which could inhibit downstream applications.
12. Transfer the Adsorption Column AC to a clean 1.5 mL microcentrifuge tube. Add 100 μL Elution Buffer EB directly onto the center of the adsorption membrane. Let it stand at room temperature for 3–5 min, then centrifuge at 13,000 rpm for 1 min to elute DNA.
13. Store the purified DNA at –20°C. For long-term storage, keep at –70°C.
Quality Control Results – DNA Concentration and Purity
|
Sample No. |
Tissue Type |
Concentration (Nanodrop, ng/μL) |
A260/A280 |
A260/A230 |
|
1 |
Mouse Skin |
69.398 |
1.71 |
1.07 |
|
2 |
Mouse Skin |
79.363 |
1.93 |
1.28 |
|
3 |
Mouse Kidney |
52.986 |
1.95 |
1.52 |
|
4 |
Mouse Kidney |
132.727 |
1.91 |
2.16 |
|
5 |
Mouse Liver |
37.771 |
2.01 |
1.35 |
|
6 |
Mouse Liver |
83.556 |
1.94 |
1.92 |
|
7 |
Mouse Lung |
32.772 |
1.93 |
1.17 |
|
8 |
Mouse Lung |
39.780 |
1.98 |
1.47 |
Quality Control Results – DNA Integrity Analysis
Conclusion
Genomic DNA was extracted from eight mouse tissue samples using Yeasen’s Hieff™ Blood/Cell/Tissue/Bacteria DNA Fast Kit (Cat. No. 18701ES). DNA concentration and purity were assessed by Nanodrop, and integrity was evaluated by agarose gel electrophoresis.
The results show that:
- DNA yields met expected requirements;
- Purity was good (A260/A280 ≈ 1.7–2.0; A260/A230 > 1.0);
- DNA integrity was high, with no obvious degradation or smearing observed on gels.
The extracted DNA is suitable for downstream applications such as PCR, qPCR, sequencing, and enzymatic reactions.