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. Transfer 2 mL of cultured bacterial suspension into a microcentrifuge tube. Centrifuge at 10,000 rpm for 30 seconds, then carefully remove and discard the supernatant as completely as possible to collect the bacterial pellet.
2. Resuspend the pellet in 200 μL of 1× PBS, centrifuge again at 10,000 rpm for 30 seconds, and discard the supernatant. Fully resuspend the cell pellet by vortexing or pipetting up and down in 180 μL of 1× PBS.
3. Add 20 μL of Proteinase K Solution, mix thoroughly. Then add 200 μL of Binding Buffer BD, and immediately vortex to ensure complete mixing. Incubate the mixture at 70°C for 10 minutes.
4. Optional step: If significant RNA contamination is expected, add 5 μL of 100 mg/mL RNase A solution before adding Binding Buffer BD, mix by vortexing, and incubate at room temperature for 5–10 minutes.
5. After cooling to room temperature, add 100 μL of isopropanol, and immediately vortex to mix thoroughly. A flocculent precipitate may form at this stage.
6. Transfer the entire mixture to an Adsorption Column AC placed in a collection tube. Centrifuge at 13,000 rpm for 1 minute, then discard the flow-through.
7. Add 500 μL of Protein Removal Buffer PL (ensure absolute ethanol has been added prior to use), centrifuge at 13,000 rpm for 30 seconds, and discard the flow-through.
8. Add 600 μL of Wash Buffer WB (ensure absolute ethanol has been added prior to use), centrifuge at 13,000 rpm for 30 seconds, and discard the flow-through.
9. Repeat step 7 once.
10. Place the Adsorption Column AC back into an empty collection tube and centrifuge at 13,000 rpm for 2 minutes to completely remove residual wash buffer. This minimizes ethanol carryover, which could inhibit downstream enzymatic reactions.
11. Transfer the Adsorption Column AC to a clean 1.5 mL microcentrifuge tube. Add 100 μL of Elution Buffer EB directly onto the center of the adsorption membrane. Let it stand at room temperature for 3–5 minutes, then centrifuge at 13,000 rpm for 1 minute to elute the DNA.
12. Store the purified DNA at –20°C. For long-term storage, keep at –70°C.
Quality Control Results – DNA Concentration and Purity
|
Sample No. |
Concentration (Nanodrop, ng/μL) |
A260/A280 |
A260/A230 |
|
1 |
369.717 |
2.07 |
2.07 |
|
2 |
391.245 |
2.08 |
2.10 |
|
3 |
341.846 |
2.10 |
2.11 |
|
4 |
382.898 |
2.05 |
2.03 |
Quality Control Results – DNA Integrity Analysis
Conclusion
Genomic DNA was extracted from four Escherichia coli samples using Yeasen’s spin-column-based 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 showed that the extracted DNA exhibited:
- Sufficient yield meeting expected requirements,
- High purity (A260/A280 and A260/A230 ratios within acceptable ranges), and
- Excellent integrity, with no obvious degradation or smearing observed on the gel.
The purified DNA is suitable for downstream applications such as PCR, sequencing, and other molecular biology assays.