產(chǎn)品介紹

The E.Z.N.A.? Soil DNA Kit allows rapid and reliable isolation of high-quality genomic DNA from various soil samples. This kit can isolate microbial DNA from yeast, fungi, and grampositive or negative bacteria that inhabit a range of samples including clay, sandy, peaty, chalky, or loamy soil samples. This kit not only includes Disruptor Tubes prefilled with glass beads for efficient sample homogenization but also features a unique inhibitor removal reagent (cHTR Reagent) for effective removal of humic acid and other PCR inhibitors from eluted DNA. The extraction methodology combines the reversible nucleic acid-binding properties of HiBind? matrix with the speed and versatility of spin column technology for DNA purification. Up to 250 mg soil samples can be processed in 60 minutes or up to 1g soil samples in 2.5 hours. Purified DNA is suitable for PCR, restriction digestion, and next-generation sequencing.

參數(shù)

組分

流程

DNA Extraction and Purification from 100–250 mg Soil

1. Add 100-250 mg soil sample and 725 μL SLX-Mlus Buffer to a Disruptor Tube. Vortex at maximum speed for 3-5 minutes to lyse samples. Centrifuge at 500g for 5 seconds to remove drops of liquid from the lid.

2. Add 72 μL DS Buffer. Vortex to mix thoroughly. Incubate at 70°C for 10 minutes. Briefly vortex the tube once during incubation. Centrifuge at 10,000g for 5 minutes at room temperature.

3. Transfer 400 μL supernatant into a new 1.5 mL microcentrifuge tube (not provided). Add 135 μL chilled P2 Buffer and 200 μL cHTR Reagent that has been completely resuspended. Vortex to mix thoroughly. Centrifuge at maximum speed for 1 minute.

4. Transfer cleared supernatant to a new 1.5 mL microcentrifuge tube. If supernatant still has a dark color from the soil, add 200 μL cHTR Reagent, vortex to mix thoroughly, and centrifuge at maximum speed for 1 minute. Transfer cleared supernatant to a new microcentrifuge tube.
   Note: This will require additional cHTR Reagent (Cat# CHTR-50) to be purchased separately.

5. Add an equal volume of XP1 Buffer. Vortex to mix thoroughly.

6. Insert a HiBind? DNA Mini Column into a 2 mL Collection Tube. Transfer up to 700 μL sample from Step 5 to the HiBind? DNA Mini Column. Centrifuge at 10,000g for 1 minute at room temperature. Discard the filtrate and reuse the Collection Tube.

7. Using the same Collection Tube, repeat Step 6 until all the lysate has passed through the HiBind? DNA Mini Column.

8. Add 500 μL HBC Buffer diluted with 100% isopropanol. Centrifuge at 10,000g for 1 minute. Discard the filtrate and Collection Tube.

9. Transfer the HiBind? DNA Mini Column into a new 2 mL Collection Tube. Add 700 μL DNA Wash Buffer diluted with 100% ethanol. Centrifuge at 10,000g for 1 minute. Discard the filtrate and reuse the Collection Tube.

10. Using the same Collection Tube, repeat Step 9 for a second DNA Wash Buffer wash step.

11. Centrifuge the empty HiBind? DNA Mini Column at maximum speed for 2 minutes at room temperature. This step is critical in removing residual ethanol that may interfere with downstream applications.

12. Transfer the HiBind? DNA Mini Column into a clean 1.5 mL microcentrifuge tube. Add 50-100 μL Elution Buffer heated to 70°C directly onto the center of HiBind? matrix. Let sit at room temperature for 1-2 minutes. Centrifuge at maximum speed for 1 minute.

13. Take the filtrate from Step 12 and place onto the center of the same HiBind? DNA Mini Column used in the procedure. Let sit at room temperature for 1 minute. Centrifuge at maximum speed for 1 minute.

14. Store eluted DNA at -20°C.

DNA Extraction and Purification from 250–1,000 mg Soil

1. Transfer the glass beads from a Disruptor Tube to a 15 mL conical tube. Add 0.25-1 g soil sample and 1 mL SLX-Mlus Buffer. Vortex at maximum speed for 3-5 minutes to lyse samples.

2. Add 100 μL DS Buffer. Vortex to mix thoroughly. Incubate at 70°C for 10 minutes. Briefly vortex the tube once during incubation. Centrifuge at 3,000g for 2 minutes at room temperature.

3. Transfer 800 μL supernatant into a new 2 mL microcentrifuge tube (not provided). Add 270 μL chilled P2 Buffer. Vortex to mix thoroughly. Let sit on ice for 5 minutes. Centrifuge at maximum speed for 10 minutes.

4. Carefully transfer the supernatant to a new 2 mL microcentrifuge tube. Add 0.7 volumes 100% isopropanol. Mix thoroughly by inverting tube for 20-30 times. (If the soil contains very low DNA, incubate the sample at -20°C for 1 hour. Centrifuge at maximum speed for 10 minutes. Carefully aspirate and discard the supernatant. Do not disturb the DNA pellet.)

5. Invert the tube on absorbent paper for 1 minute to drain the liquid. Add 200 μL Elution Buffer. Vortex for 10 seconds. Incubate at 70°C for 10 minutes. Vortex briefly. Centrifuge at maximum speed for 1 minute.

6. Add 100 μL cHTR Reagent that has been completely resuspended. Vortex to mix thoroughly. Let sit at room temperature for 2 minutes. Centrifuge at maximum speed for 2 minutes.

7. Transfer the cleared supernatant to a new 1.5 mL microcentrifuge tube. If supernatant still has a dark color from the soil, repeat Step 6 for a second cHTR Reagent step. This will require additional cHTR Reagent (Cat# CHTR-50) to be purchased separately.

8. Add an equal volume of XP1 Buffer to mix thoroughly.

9. Insert a HiBind? DNA Mini Column into a 2 mL Collection Tube. Transfer the sample from Step 8 to the HiBind? DNA Mini Column. Centrifuge at 10,000g for 1 minute at room temperature. Discard the filtrate and reuse the Collection Tube.

10. Add 500 μL HBC Buffer diluted with 100% isopropanol. Centrifuge at 10,000g for 1 minute. Discard the filtrate and the Collection Tube.

11. Transfer the HiBind? DNA Mini Column into a new 2 mL Collection Tube. Add 700 μL DNA Wash Buffer diluted with 100% ethanol. Centrifuge at 10,000g for 1 minute. Discard the filtrate and reuse the Collection Tube.

12. Centrifuge the empty HiBind? DNA Mini Column at maximum speed for 2 minutes at room temperature. This step is critical in removing residual ethanol that may interfere with downstream applications.

13. Transfer the HiBind? DNA Mini Column into a clean 1.5 mL microcentrifuge tube. Add 50-100 μL Elution Buffer heated to 70°C directly onto the center of HiBind? matrix. Let sit at room temperature for 1-2 minutes. Centrifuge at maximum speed for 1 minute.

14. Take the filtrate from Step 13 and place onto the center of the same HiBind? DNA Mini Column used in the procedure. Let sit at room temperature for 1 minute. Centrifuge at maximum speed for 1 minute.

15. Store eluted DNA at -20°C.