ALFA-Soil DNA Extraction Mini Kit

Product Introduction

ALFA-Soil DNA Extraction Mini Kit is a kit designed for the extraction of trace DNA in high-impurity soil. This product adopts silica gel column purification technology, which is suitable for extracting high-yield and high-purity total DNA from various soil samples such as conventional soil, sediment, heavy metal contaminated soil, desert dry soil, etc. Combined with the unique two-step impurity removal technology, It can efficiently adsorb humic acid and other inhibitors in soil samples. This kit has simple steps, can complete the extraction and purification of trace DNA within 1 hour, without liquid nitrogen grinding, phenol chloroform or other reagents, safe, fast and reliable. The purified DNA can be directly used in experiments such as PCR, Southern Blot, enzyme digestion and next-generation sequencing.

Product Components

Storage Conditions and Expiration Date

This product can be stored at room temperature (15~25°C) for 12 months, and should be stored at 2~8°C for long-term storage.

At low temperature, ALFA Buffer Lysis B and ALFA Binding Buffer may form precipitates. The 37 ℃ water bath can completely dissolve the precipitates without affecting the effect of use.

Prepared by User

Vortexer or bead mill, water bath or metal bath, absolute ethanol, centrifuge.

Preparations

1. ALFA Washing Buffer

2.  Before use, add absolute ethanol to dilute as indicated on the bottle label.

Experimental Procedure

1. Homogeneous lysis of soil (choose the grinding method according to the actual situation, and preferably choose the bead mill)

Method 1: In Glass Beads Tube I, first add 0.25g soil sample and 750 μL ALFA Lysis Buffer A, vortex on the vortexer at the highest speed for 5~10 seconds, then add 60 μL ALFA Lysis Buffer B to the sample, After vortexing for 10 minutes, proceed to step 2.

Method 2: Add 0.25g of soil sample to Glass Beads Tube I, first add 750 μL ALFA Lysis Buffer A and then add 60 μL ALFA Lysis Buffer B; (ALFA Lysis Buffer A and ALFA Lysis Buffer B cannot be premixed) homogenate in the bead mill. Different bead mills need to set parameters according to the intensity due to difference in powers (for example: when using FastPrep-24® (MP), the recommended speed is 8.0 and the time is 45 s).

Note 1: Pre-added ALFA Lysis Buffer A can prevent nucleic acid degradation when the sample is kept at room temperature for a long time.

2. Take out the grinding tube and place it in a water bath at 55°C for 15 minutes; (Alfa PI Buffer 1 and ALFA PI Buffer B can be prepared separately in advance for the water bath time)

3. Take out the grinding tube and centrifuge at 12,000 g for 2 min;

4. Transfer all the supernatant to a new 2mL centrifuge tube, add 250μL of ALFA PI Buffer 1 to the supernatant, and vortex to mix.

5. Add 200 μL of ALFA PI Buffer B to the sample, shake well or vortex the sample at 15 gears until it becomes milky, and place it at 4°C for 10 min; (use a 2ml centrifuge tube for splitting a single tube, and mix the samples in multiple tubes at the same time into 15ml centrifuge tube, use a 50ml centrifuge tube for more than six tubes)

Note 2: The reagents should be added to the sample in sequence with ALFA PI Buffer 1 at first and then ALFA PI Buffer B, they cannot be added to the centrifuge tube at the same time in advance.

Note 3: If the sample is divided into n tubes, the amount of ALFA PI Buffer 1, ALFA PI Buffer B, and ALFA Binding Buffer will increase n times in the same proportion.

6. Centrifuge at 12,000 x g for 5 min at room temperature;

7. Transfer the supernatant to a new centrifuge tube;

8. Add the equal volume of ALFA Binding Buffer to the centrifuge tube and mix by inversion.

9. Pack the gDNA Extraction Mini Columns in a 2ml collection Tube. Transfer 700 μl of the mixture to the column. Centrifuge at 12,000 x g for 1 min. Discard the effluent and place the column back into the collection tube.

10. Centrifuge the remaining mixture as above until the transfer was complete.

Note 4: When transferring the mixture to the column, the volume should not exceed 700 μl each time.

11. Add 600 μL of ALFA Washing Buffer2 diluted in ethanol to the column. After standing for 5 minutes, centrifuge at 12,000 x g for 1 minute. Discard the effluent and put the column back into the collection tube;

12.Repeat the above steps once;

13.Dry the column by centrifugation at 13.12,000 x g for 2 min.

14. Pack the column in a 1.5ml centrifuge tube. Add 35-50 μl of Buffer EB preheated to 65°C to the center of the membrane of the column, and leave it at room temperature for 3 minutes. Centrifuge at 13,000 x g for 1 min.

15. Discard the DNA binding column. Store DNA at -20ºC.

Common Issues

1. DNA has color

Excessive starting sample: For soil samples rich in humic acid, such as forest soil and grassland soil, it is recommended to reduce the sample amount by half (~0.25 g).

Lysis Buffer B has precipitate released: recommended to incubate at 37°C and use it after the precipitate is fully dissolved.

2. Low DNA yield

Low DNA content in soil: The nucleic acid content of soil samples is low. You can try to combine the nucleic acids extracted for multiple times, and then concentrate and purify them.

Low impurity content in the sample: For example, some desert soils have low impurity content such as humic acid, so steps 5-6 can be omitted, and the DNA yield can be improved by directly transferring from step 4 to step 7.

Low elution efficiency: Poor water solubility due to large genomic DNA fragments. It is recommended to increase the elution volume and the number of elution times to improve DNA yield.

Incorrect reagent preparation: ALFA Wash Buffer 2 must be diluted with the correct volume of absolute ethanol according to the label of the reagent bottle or the instructions.

Excessive starting sample: The amount of soil is too large, resulting in insufficient remaining space in the homogenization tube, and the ideal vortex effect cannot be achieved. It is recommended to reduce the sample volume or change to a 4 mL centrifuge tube for homogenization.

Insufficient lysis: It is recommended to use a bead mill instead of manual vortexing, or adjust the speed of the vortexer to the highest speed during manual vortexing, and vortex continuously for 5-10 minutes.

3. Serious DNA fragmentation

Manual vortexing takes too long: It is recommended to use a bead mill instead of manual vortexing to reduce DNA fragmentation.

Excessive starting sample: For soil samples rich in humic acid, such as forest soil and grassland soil, it is recommended to reduce the sample amount by half (~0.25g).

Excessive vortexing or shaking time: It is recommended to optimize bead milling conditions to reduce vortexing or shaking time to reduce DNA fragmentation.

4. RNA Contamination

RNase digestion: RNase A can be added to the lysate to digest to remove RNA contamination.

5. Abnormal ratio of OD260/280 or OD260/230

RNA contamination: RNase A can be added to the lysate for digestion to remove RNA contamination.

The nucleic acid concentration is too low: the nucleic acid concentration is too low, resulting in a large deviation of the OD ratio.

Ethanol is not added to Washing Buffer 2 or the amount added is not enough: Add the correct volume of absolute ethanol according to the label of the reagent bottle or the instructions.