| M666121 | Component | 96 T | Storage | | M666121A | Buffer KCL | 80 mL | RT | | M666121B | Buffer CW1 (concentrate) | 80 mL | RT | | M666121C | Buffer GW2 (concentrate) | 26 mL | RT | | M666121D | Buffer MW3 | 80 mL | RT | | M666121E | Buffer EB | 30 mL | RT | | M666121F | Proteinase K | 2脳25 mg | RT | | M666121G | Proteinase K Storage Buffer | 2脳1.25 mL | RT | | M666121H | Magbeads PN | 2脳1 mL | RT |
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Product Introduction
This kit provides a simple, fast, and efficient saliva DNA extraction method, suitable for extracting genomic DNA from saliva. In the presence of high salt, DNA binds to the surface of silica coated Magheads. After rinsing, high-purity DNA is eluted in Buffer EB or deionized water. The purified DNA has good purity (A260/280 ratio between 1.7-1.9) and high integrity (>15 kb), and can be used for downstream experiments such as second-generation sequencing, quantitative PCR, and chip detection.
Self provided instruments and reagents
1) Constant temperature mixer
2) 2/15 ml magnetic frame
3) 32 channel nucleic acid extractor
4) 96 channel nucleic acid extractor
5) 96 DW Plate
6) 8 channel Comb
7) Spin tips pack
8) Anhydrous ethanol Preparation and important precautions before the experiment
1. Add 1.25 ml Protein K Storage Buffer to 25mg Protein K to dissolve it, then store at -20 ℃. The prepared Protein K solution should not be left at room temperature for a long time to avoid repeated freeze-thaw cycles, which may affect its activity.
2. Before the first use, add anhydrous ethanol to Buffer GW1 and Buffer GW2 according to the label of the reagent bottle and mark them properly.
3. Magheads are strictly prohibited from freezing or centrifugation. Freezing and centrifugation may cause irreversible damage to Magheads.
Operation steps
I. Manual single tube operation
1. Add 20 to a 2.0 mL centrifuge tube in sequence μ Protease K, 350 μ Saliva and 500 μ Buffer KCL.
2. After vortex shaking and mixing, place the centrifuge tube on a constant temperature mixer at 65 ℃ and 1600 rpm to shake and crack for 20 minutes, forming a cracking solution.
Attention: If there is no constant temperature mixer, vortex the centrifuge tube for 10 seconds and incubate it in a 65 ℃ water bath for 20 minutes every 5 minutes The clock vortex oscillates for 10 seconds.
3. Remove the centrifuge tube from the constant temperature mixer, centrifuge briefly, and then add 20 μ Magheads PN. Afterwards, place the centrifuge tube on a constant temperature mixer at 25 ℃ and 1600 rpm, shake and crack for 10 minutes, or invert the centrifuge tube and mix continuously for 15 minutes.
4. Place the centrifuge tube on a magnetic stand and let it stand for 1 minute. After Magheads are completely adsorbed on the side wall of the centrifuge tube, discard the solution thoroughly (keep the centrifuge tube fixed on the magnetic stand).
5. Remove the centrifuge tube from the magnetic frame and add 750 μ Buffer GW1 (please check if anhydrous ethanol has been added before use), vortex point shake for 1 minute or vortex shake for 5 seconds, then place it on a constant temperature mixer at 25 ℃ and 1600 rpm, shake and mix for 2 minutes (ensure that Magheads are in a mixed state during the shaking process). Afterwards, place the centrifuge tube on a magnetic stand and let it stand for 1 minute. After Magheads are completely adsorbed on the side wall of the centrifuge tube, gently invert the magnetic stand and wash the impurities on the centrifuge tube cover to completely discard the solution (keep the centrifuge tube fixed on the magnetic stand).
6. Repeat step 5.
7. Remove the centrifuge tube from the magnetic frame and add 750 μ Buffer GW2 (please check if anhydrous ethanol has been added before use), vortex point shake for 1 minute or vortex shake for 5 seconds, and then place it on a constant temperature mixer at 25 ℃ and 1600 rpm to shake and mix for 2 minutes (ensure that Magheads are in a mixed state during the shaking process). Afterwards, place the centrifuge tube on a magnetic stand and let it stand for 1 minute. After Magheads are completely adsorbed on the side wall of the centrifuge tube, gently invert the magnetic stand and wash the impurities on the centrifuge tube cover to completely discard the solution (keep the centrifuge tube fixed on the magnetic stand).
8. Keep the centrifuge tube fixed on the magnetic frame, add 750ul Buffer MW3 to the centrifuge tube, and immediately discard the solution thoroughly after the suspended magnetic beads reattach to the magnetic frame.
9. Keep the centrifuge tube fixed on the magnetic frame, use a pipette to further remove the solution from the bottom and cover of the centrifuge tube, and then leave it at room temperature for 5-10 minutes to allow the ethanol to evaporate completely. 10. Remove the centrifuge tube from the magnetic frame and add 50-200 μ Buffer EB. Vortex oscillation causes the magnetic beads to completely suspend in the eluent and then place them on a constant temperature mixer at 56 ℃ and 1600 rpm for 10 minutes of shaking and elution, or incubate the centrifuge tube in a 56 ℃ water bath for 10 minutes, with vortex oscillation every 3 minutes for 10 seconds.
11. Place the centrifuge tube on a magnetic stand and let it stand for 2 minutes. After Magheads are completely adsorbed on the side wall of the centrifuge tube, transfer the eluent to a new centrifuge tube using a pipette and store at -20 ℃ for later use. II. Matching with a 32 channel nucleic acid extractor
1. Add the corresponding reagents to the 96DW deep well plate according to the table below | Position | Reagent | | 1&7 Colume | Proteinase K: 20 μl
Sample: 350 μl
Buffer KCL: 500 μl
Magbeads PN: 20 μl | | 2&8 Colume | Buffer GW1: 750 μl | | 3&9 Colume | Buffer GW1: 750 μl | | 4& 10 Colume | Buffer GW2: 750 μl | | 5& 11 Colume | Buffer MW3: 750 μl | | 6& 12 Colume | Buffer EB: 100 μl |
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4. Place the deep well plate and magnetic sleeve that have been added to the reagent at the corresponding positions on the 32 channel nucleic acid extractor, run the saliva extraction program, and after about 28 minutes, the program ends. Remove the deep well plate and magnetic sleeve.
5. Transfer the elution products from columns 6 and 12 of the deep well plate to a 1.5 ml centrifuge tube for low-temperature storage. III. Matching with 96 channel nucleic acid extractor
1. Add the corresponding reagents to the 96DW deep well plate according to the table below | Position | Reagent | | Plate 1 | Proteinase K: 20 μl
Sample: 350 μl
Buffer KCL: 500 μl
Magbeads PN: 20 μl | | Plate 2 | Buffer GW1: 750 μl | | Plate 3 | Buffer GW1: 750 μl | | Plate 4 | Buffer GW2: 750 μl | | Plate 5 | Buffer MW3: 750 μl | | Plate 6 | Buffer EB: 100 μl |
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2. Place the deep well plate and magnetic sleeve that have been added to the reagent at the corresponding positions on the 96 channel nucleic acid extractor, run the saliva extraction program, and after about 28 minutes, the program ends. Remove the deep well plate and magnetic sleeve.
3. Transfer the elution products from Plate 6 to a 1.5 ml centrifuge tube for low-temperature storage.
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