01/Product introduction

This kit is based on the principle of silicon-based magnetic beads to specifically adsorb DNA, enzymatically releases blood genome, binds DNA molecules in a specific high-efficiency lysis buffer system, changes the environmental ionic strength, and realizes the separation and purification of genomic DNA. The genome can be efficiently extracted from 100-250ul blood samples, and the obtained genomic DNA is suitable for downstream experiments such as PCR reaction, enzyme digestion reaction and Southern hybridization.

02/Product advantage

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

2. Compatible with fresh, frozen and anticoagulated blood samples (EDTA anticoagulation, heparin anticoagulation and citrate anticoagulation)

3. Easy and quick operation, good 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/Quality inspection examples

DNA purification of the following samples: 200 μl EDTA, EDTANaF, heparin sodium, sodium citrate and other human anticoagulant blood treated with different treatments, 2 ml of the buffy coat separated from the blood, 10 μl chicken blood and 200 μl mouse blood, M: TIANGEN Marker D15000. The elution volume was 100 μl, and the agarose gel electrophoresis loading volume was 4 μl.

Analysis of experimental results: This kit has a wide range of sample applicability, and can extract high-quality DNA from human anticoagulant blood of different treatments and blood of different species.

07/Notes

1. Before using for the first time, add the corresponding volume of absolute ethanol to BufferW1 and BufferW2.

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

3. If precipitation occurs after the solution has been placed for a period of time, it can be dissolved in a 37°C water bath without affecting the effect.

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

5. Repeated freezing and thawing of blood samples will cause the extracted DNA fragments to be smaller and the extraction volume to decrease. The resulting genomic DNA should also avoid repeated freezing and thawing as much as possible to avoid degradation. If extracting genomic DNA from frozen blood, it is recommended to use a 37°C water bath to quickly thaw before proceeding with subsequent operations.

6. Storage of blood samples:
1) Short-term storage: blood samples that have been added with anticoagulants can be stored at 2～8℃ for up to 10 days. For some experiments such as Southern hybridization, you need to obtain complete and full-length genomic DNA. Please place blood samples at 2～8. Store at ℃ for no more than 3 days, at which time the degree of degradation of genomic DNA is relatively light.
2) Long-term storage: The blood that has been added with anticoagulant should be stored at -70°C (if high molecular weight DNA is extracted, EDTA is recommended as an anticoagulant).

08/How to use

1. Take 100-250ul blood in a 1.5ml clean centrifuge tube.

2. Add 300ul Buffer KA and 20ul Proteinase K to it, vortex and mix for 30 seconds.

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

4. Add 200ul of isopropanol and vortex for 20s and add 20ul of 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 W1, vortex and mix for 2 minutes, perform magnetic separation, and remove the liquid other than the magnetic beads (Caution: Do not suck off the magnetic beads).

9. Repeat step 8 once.

10. Remove the centrifuge tube from the magnetic stand, add 600ul Buffer W2, vortex and mix for 2 minutes, perform magnetic separation, and remove 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 100-200ul Buffer TE, pipette and mix 20 times, incubate at 65°C for 8 min, and mix twice during the period.

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°C.

09/Common problems and coping strategies

◆The purity of genomic DNA obtained is low

1. It is recommended to increase the elution volume of Buffer W1 and Buffer W2 by 100ul.

2. Use fresh blood 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.

4. Before eluting with Buffer TE, it was not fully dried, and a small amount of ethanol remained.

◆Low concentration of genomic DNA obtained

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

2. Reduce the amount of Buffer TE or extend the incubation time at 65°C 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 10ul 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.

◆Agglomeration 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. .