1. Components of the reagent kit
| Specifications | 50T | 100T |
| Cat. No. | SN0219 | SN0220 |
| DNA Extraction Columns (set) | 50 (set) | 100 (set) |
| Reagent Buffer B | 20 ml | 2 × 20 ml |
| Reagent Buffer C | 30 ml | 2 × 30ml |
| 10×Red Blood Cell Lysis Buffer | 60 ml | 2 × 60ml |
| Wash Buffer 1 | 15 ml | 2 × 15 ml |
| Elution Buffer | 20 ml | 20 ml |
| Proteinase K | 1ml | 2x1ml |
| RNaseA | 1ml | 2x1ml |
| Instruction Manual | 1 | 1 |
2. Storage
This reagent kit should be stored at room temperature (15-25℃) and in dry conditions, with a shelf life of 12 months. The DNA extraction purification columns can be stored for 1 year in a cool and dry environment. Proteinase K and RNase A contain preservatives, allowing transportation at room temperature, but for long-term storage, they should be kept at -20℃.
3. Instructions for Using the Reagent Kit
3.1 This reagent kit is intended for molecular biology research and should not be used for disease diagnosis or treatment.
3.2 Some components in the reagent kit contain irritants. Protective measures such as wearing protective clothing and goggles are recommended.
3.3 During the usage of this reagent kit, a high-speed centrifuge, water bath (metal bath), vortex mixer, anhydrous ethanol, sterile deionized water, and EP tubes need to be prepared by the user.
4. Introduction to the Reagent Kit
The Blood DNA Purification Reagent Kit offers a rapid and effective method for purifying DNA from blood and other bodily fluids. By lysing red blood cells with a red blood cell lysis buffer, DNA can be efficiently collected.
The Blood DNA Rapid Purification Kit can extract total DNA from whole blood (including blood, serum, plasma, and other bodily fluids) within 30 minutes. The entire purification process doesn’t require toxic reagents such as phenol-chloroform. The extracted DNA can be directly used for PCR, Southern blotting, and other applications.
5. Experimental Principles and Procedures
6. Extraction Process
Before Starting the Experiment:
A. Red Blood Cell Lysis Buffer 1x Preparation: Dilute the 10x Red Blood Cell Lysis Buffer to 1x volume using RNase-free water.
B. Reagent Buffer B and Reagent Buffer C may precipitate at low temperatures. It is recommended to heat at 65℃ for 5 minutes to dissolve the precipitate before use.
C. Prior to using Wash Buffer 1, add the specified amount of anhydrous ethanol as indicated on the reagent bottle label, and mark a check on the label to indicate ethanol addition.
D. Elution Buffer is a 0.1x TE solution with minimal EDTA. If EDTA might affect subsequent experiments, it is suggested to substitute Elution Buffer with sterile deionized water.
- Sample Handling: (Collect 0.1-5 ml blood samples)
A. Add 2-3 times the volume of 1x Red Blood Cell Lysis Bufferto the blood or sample (Dilute the Red Blood Cell Lysis Buffer 10x to 1x before use), mix thoroughly, centrifuge at 12,000 rpm for 1 minute, carefully remove the supernatant. The precipitate should theoretically be white or pale red. Add Reagent Buffer B to the precipitate (for 0.1-1ml blood samples, add 200μLReagent Buffer B; for 1-2ml blood samples, add 400μL Reagent Buffer B).
B. If handling samples from low-level organisms like poultry or birds with nucleated red blood cells, a smaller sample volume is needed. In such cases, omit the 1x Red Blood Cell Lysis Buffer and directly add 400μLof Reagent Buffer B and 20μL of RNaseA (10 mg/ml), mix thoroughly, and incubate at room temperature for 5 minutes.
2. Add 10μLRNaseA (10 mg/ml), 20μL Proteinase K (10 mg/ml), mix thoroughly, digest at 65℃ for 10 minutes. During this period, invert and mix 2-3 times until digestion is complete.
3. Add 200μLof Reagent Buffer C to the lysate, mix by pipetting, add 200μL of anhydrous ethanol, mix by pipetting.
4. Apply the obtained liquid to the DNA extraction purification column (set) (approximately 650~700μl each time), centrifuge at 12,000 rpm for 30 seconds, discard the collected waste, and re-insert the collection tube into the DNA extraction purification column (set) for the next step.
5. Add 700μLof inhibition removal buffer, centrifuge at 12,000 rpm for 30 seconds, discard the waste.
6. Place the DNA extraction purification column (set) in a new collection tube, add 500μLof Wash Buffer 1, centrifuge at 12,000 rpm for 30 seconds, discard the waste, and re-insert the DNA extraction purification column (set) into the collection tube for the next step.
(Note: Ensure anhydrous ethanol has been added to Wash Buffer 1.)
7. Repeat step 6.
8. Place the DNA extraction purification column (set) into a new centrifuge tube, uncover, incubate at 65℃ in a water bath for 2 minutes. Extend this step appropriately to evaporate ethanol as much as possible to prevent ethanol residue from affecting downstream experiments.
9. Suspend 50-100μLof Elution Buffer onto the membrane of the column, centrifuge at 12,000 rpm for 1 minute, and collect the DNA.
(Note: 1. Eluting DNA with 50μL of Elution Buffer can increase DNA concentration but reduces total DNA yield; 2. The eluted DNA wash can be reapplied to the DNA extraction purification column. Centrifuge at 12,000 rpm for 1 minute again to increase DNA yield.
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