16399712922.pdf

01/Product introduction

This kit is based on the principle of specific adsorption of DNA by silicon-based magnetic beads, combined with chemical and enzymatic methods to release fecal genomes, and a specially developed buffer system to efficiently remove polysaccharides, lipids, acids, digestive enzymes and bile red The genomic DNA molecules can be stably extracted from stool samples with a volume of 0.25-5g. The obtained genomic DNA is suitable for downstream experiments such as conventional PCR reaction, qPCR reaction, enzyme digestion reaction and detection.

02/Product advantage

1. No need for toxic reagents such as phenol or chloroform

2. Compatible with manual and high-throughput operation

3. Easy and quick operation, stable quality

03/Product content

04/Storage conditions

It is valid for 1 year when stored at room temperature.

05/Bring your own reagents

Isopropanol, absolute ethanol

06/Notes

1. Before using for the first time, add the corresponding volume of absolute ethanol to Buffer FW2 and Buffer FW3.

2. Shake the solution in each bottle before each use to make the system uniform and ensure the extraction effect.

3. If precipitation (such as Buffer KE and Buffer KEG) occurs after the solution is left for a period of time, it can be dissolved in a 37¡ãC water bath without affecting the effect.

4. Use sterile centrifuge tubes and pipette tips to avoid contamination by exogenous nucleases.

5. If the single-time fecal extraction volume is greater than 0.5g, the addition volume and elution volume of the system can be expanded proportionally.

07/How to use

1. Take 0.25-0.5g of feces into a 2ml clean centrifuge tube.

2. Add 500ul Buffer KE and 20ul Proteinase K to it, vortex and mix for 30 seconds.

3. Incubate at 68¡ãC (both water bath and metal bath) for 10 minutes, vortex and mix for 5 seconds every 2-3 minutes.

4. Centrifuge at 12000rpm or 13400g for 3min at room temperature, transfer the supernatant to a 1.5ml centrifuge tube, and add 300ul Buffer KEG

Mix with 200ul isopropanol and 20ul magnetic bead suspension (note: vortex and mix the magnetic bead suspension before adding).

5. Vortex and mix for 30 seconds, and let stand for 1 minute.

6. Repeat step 5 twice.

7. Place the transfer centrifuge tube on the magnetic stand. After magnetic separation, carefully suck off the liquid (Caution: Do not suck off the magnetic beads).

8. Remove the centrifuge tube from the magnetic stand, add 800ul Buffer FW1, vortex and mix for 30 seconds, let it stand at room temperature for 1 min, perform magnetic separation, and remove the liquid other than the magnetic beads (Caution: Do not suck off the magnetic beads).

9. Remove the centrifuge tube from the magnetic stand, add 800ul Buffer FW2, vortex and mix for 30 seconds, let it stand at room temperature for 1 min, perform magnetic separation, and remove liquid other than the magnetic beads (Caution: Do not suck off the magnetic beads).

10. Remove the centrifuge tube from the magnetic stand, add 800ul Buffer FW3, vortex and mix for 30 seconds, let it stand at room temperature for 1 min, perform magnetic separation, and remove the liquid other than the magnetic beads (Caution: Do not suck off the magnetic beads).

11. Repeat step 10 once.

12. Transfer the centrifuge tube to a magnetic rack and dry it at room temperature for 8-10 minutes (Note: the drying time should not exceed 10 minutes, otherwise it will affect the genome quality).

13. Remove the centrifuge tube, add 50-150ul Buffer FTE, vortex and mix for 30 seconds or pipette to mix 20 times, incubate at 65¡ãC for 8 minutes, vortex and mix for 5 seconds or pipette and mix for 2 times.

14. The transfer centrifuge tube is placed on a magnetic stand for magnetic separation, and the liquid except the magnetic beads is transferred to a new centrifuge tube and stored at -20¡ãC.

08/Common problems and coping strategies

¡ôThe purity of genomic DNA obtained is low

1. It is recommended to increase the elution volume of Buffer FW1 and Buffer FW2 by 100ul.

2. Use fresh stool samples for extraction.

3. During the washing process, the magnetic beads were not sufficiently dispersed, and there was protein aggregation. It is recommended to increase the strength and time of blowing or vortex mixing or reduce the amount of isopropanol from 200ul to 100ul.

4. The amount of extracted sample is not suitable, can be appropriately enlarged or reduced

5. Before elution with Buffer FTE, it was not fully dried, and a small amount of ethanol remained.

¡ôLow concentration of genomic DNA obtained

1. It is recommended to replace with fresh samples for extraction.

2. Reduce the amount of Buffer FTE, or extend the incubation time at 65¡ãC to 15min.

3. During the elution process, if the magnetic beads are not sufficiently magnetically separated, the liquid will be transferred, resulting in the loss of magnetic beads.

4. Increase the magnetic beads by 5ul to increase the adsorption strength, thereby increasing the yield.

5. If the drying time exceeds 10 minutes, the magnetic beads are too dry and the elution efficiency is reduced.

6. In the process of elution with Buffer TE, the magnetic bead clumps were not sufficiently broken up, resulting in insufficient genome release.

¡ôAgglomeration of magnetic beads occurred during the extraction process

In the magnetic bead extraction process, because a large amount of chromatin nucleic acid is adsorbed on the surface of the magnetic beads, the phenomenon of magnetic beads agglomerates in most cases. This phenomenon can be used as an indicator of nucleic acid content. Generally speaking, clumping indicates that the sample is good and the adsorption process is smooth and efficient. As long as the operator fully breaks up the clumps during the washing and elution process, an ideal genomic DNA molecule can be obtained. .

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