Given that mangrove soil has a higher mineral and carbon content, should I try using half SL1 and half SL2 + SX (350 ul, 350 ul, 150 ul) instead? According to the manual, SL1 + SX is recommended for mineral-rich soils, while SL2 is better for soils with high organic carbon. Would a combination of both be more effective for my sample?

1. Preparation of Sample:

Approximatly 350 mg soil sample was accurately weighed and added to an MN Bead Tube Type A containing glass beads. To reduce salt and other unwanted debris, the soil was washed with 8.5% autoclaved NaCl solution and subsequently centrifuged at 11,000 x g for 2 minutes and 10 seconds.

2. Discarding Supernatant:

The supernatant was carefully discarded to avoid any loss of pellet or sample content. Meanwhile, the lysis buffers (SL1 and SL2) were pre-warmed at 65°C for 15 minutes to enhance the efficiency of cell lysis, especially for microbial cells bound to soil particles. After pre-warming, a total of 700 µL of lysis buffer (consisting of 350 µL of SL1 and 350 µL of SL2) was added to the bead tube along with 150 µL of SX buffer, ensuring the complete saturation of the sample.

3. Homogenization:

The bead tube containing the sample was subjected to vigorous vortexing for 7 minutes, followed by a 30-second pause, and then vortexed again for another 7 minutes to ensure proper mechanical disruption of microbial cells and soil particles. This step was crucial for maximizing DNA yield from complex soil samples.

4. Centrifugation:

Following homogenization, the tube was centrifuged at 11,000 x g for 2 minutes and 10 seconds. The resulting supernatant (containing lysed DNA) was carefully transferred to a new microcentrifuge tube, ensuring no bead or soil residue was carried forward.

5. Sample Lysis:

To further break down any remaining cell components or contaminants, 150 µL of SL3 Sample Lysis Buffer was added to the supernatant. The mixture was vortexed for 5 seconds and incubated at 4°C for 15 minutes to enhance lysis efficiency and improve DNA recovery. Following incubation, the tube was centrifuged again at 11,000 x g for 1 minute and 10 seconds.

6. Filtration:

The clear supernatant obtained from Step 5 was carefully loaded onto a red-filtered filter (NucleoSpin® Inhibitor Removal Column) in two portions:

• The first 700 µL of supernatant was loaded and centrifuged at 11,000 x g for 1 minute and 10 seconds. The flow-through was collected.
• The remaining supernatant was then loaded and centrifuged again for 1 minute to ensure maximum recovery.
• The combined flow-through was collected in a clean microcentrifuge tube.

7. Adjusting Binding Conditions:

To adjust the DNA binding conditions, 250 µL of Buffer SB (Binding Buffer) was added to the combined flow-through. The tube was briefly vortexed for 5 seconds to mix the binding buffer with the DNA-containing solution.

8. Loading Sample onto Column:

A NucleoSpin® Soil Column (with a green ring) was placed in a 2 mL Collection Tube. The mixed solution was divided into two parts and loaded in portions:

• 550 µL of the solution was loaded onto the column and centrifuged at 11,000 x g for 1 minute and 10 seconds.
• The flow-through was discarded, and the remaining sample was loaded again and centrifuged under the same conditions.
• This step was critical to maximize DNA retention onto the silica membrane.

9. Washing the Column:

To remove any remaining inhibitors or contaminants, a series of wash steps were performed:

• 500 µL of Buffer SB (Binding Buffer) was added.
• Centrifugation at 11,000 x g for 35 seconds was performed.
• The flow-through was discarded.

• 550 µL of Buffer SW1 was added.
• Centrifugation at 11,000 x g for 35 seconds was done.
• The flow-through was discarded.

• 650 µL of Buffer SW2 was added.
• The lid was closed, the column was briefly vortexed for 2 seconds, and then centrifuged at 11,000 x g for 35 seconds.
• The flow-through was discarded.

• Another 650 µL of Buffer SW2 was added, the column was vortexed for 2 seconds, and centrifuged again for 35 seconds.
• The flow-through was discarded.

10. Drying the Silica Membrane:

To ensure no residual ethanol was present, the column was centrifuged at 11,000 x g for 2 minutes and 10 seconds. After centrifugation, I also allowed the column to air dry for 5 minutes to ensure complete membrane dryness.

11. Elution of DNA:

For elution, 40 µL of nuclease-free water was added directly onto the middle of the silica membrane without disturbing the membrane. The column was incubated at room temperature for 7 minutes with the closed led, allowing water to diffuse through the membrane.

• The tube was centrifuged at 11,000 x g for 35 seconds, and the elution was collected in a clean microcentrifuge tube.
• This step was repeated once more to maximize DNA recovery, resulting in approximately 70-80 µL of total elution volume.