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Blood gDNA Isolation Mini Kit(Solution)

Packing Specification：

Product Introduction:
Based on the characteristics of whole blood, this kit extracts genomic DNA through several rapid steps: first, Red Blood Cell Lysis Buffer lyses and removes DNA-free red blood cells; then Nucleus Lysis Buffer lyses white blood cells to release genomic DNA; subsequently, Protein Precipitation Buffer selectively precipitates and removes proteins; finally, pure genomic DNA is precipitated with isopropanol and redissolved in DNA Dissolution Buffer. The operation is simple, with low experimental cost, high yield, and good purity, making it suitable for various downstream experiments.

Application Scope:
Suitable for the rapid extraction of small-volume whole blood genomic DNA.

Product Features:
1.Optimized red blood cell lysis buffer formula selected from dozens of formulations, ensuring rapid and complete lysis.
2.No need for toxic reagents such as phenol.
3.Fast and simple; operation for a single sample can generally be completed within 30 minutes.
Stable results and high yield: typically, 4-15 µg of DNA can be extracted from 300 µL of whole blood, with a typical OD260/OD280 ratio of 1.7-1.9 and a length of 50 kb-150 kb. The DNA can be directly used for library construction, PCR, Southern blot, and various restriction enzyme digestion reactions.

Operation Steps:
This kit is solution-based and can process blood volumes scaled up or down in proportion (20 µL-10 mL).
1.Pipette 900 µL of 1x Red Blood Cell Lysis Buffer (dilute 10× stock to 1x with deionized water before use) into a 1.5 mL centrifuge tube.

2.After bringing anticoagulated whole blood to room temperature and inverting to mix, pipette 300 µL into the centrifuge tube containing Red Blood Cell Lysis Buffer from the previous step. Invert 6-8 times and gently tap the tube wall while inverted to ensure thorough mixing.

3.Incubate at room temperature for 10 minutes (invert and tap the tube several times during incubation to facilitate red blood cell lysis).

4.Centrifuge at 12,000 rpm for 1 minute. Discard the red supernatant and invert the centrifuge tube on absorbent paper to remove residual blood from the tube mouth.
Optional step: Add another 300 µL of 1x Red Blood Cell Lysis Buffer, invert several times, incubate for 1-2 minutes, centrifuge at 12,000 rpm (2,500 x g) for 1 minute, then discard the red supernatant.

5.Use a 1 mL blue pipette tip to add 300 µL of preheated (50℃) Nucleus Lysis Buffer to the tube, and pipette up and down 10-15 times to lyse the white blood cells until the solution becomes clear. Do not pipette too vigorously to avoid shearing genomic DNA or generating bubbles. Then incubate in a 60-65℃ water bath or oven for approximately 10-20 minutes until the solution is clear.
Note: If there are red clotted blood clots at the bottom that cannot be dispersed, add 10 µL of Proteinase K (20 mg/mL) solution, immediately vortex to mix well, and incubate in a 50℃ incubator for approximately 30-60 minutes until the solution is clear.

6.Optional step (generally not required): Add RNase A (10 mg/mL) to the lysate to a final concentration of 30 μg/mL, invert 25 times to mix, and incubate at 37℃ for 15 minutes to remove residual RNA. Then cool to room temperature.

7.Remove the centrifuge tube from the water bath or oven, add 100-150 µL of Protein Precipitation Buffer, and vortex continuously at high speed for 25 seconds. Small protein clumps should be visible after mixing. (For Steps 1-4: Add 150 µL of Protein Precipitation Buffer for one round of 1x Red Blood Cell Lysis; add only 100 µL for two rounds of lysis.)

8.Centrifuge at 13,000 rpm for 5 minutes. A dark brown protein pellet should be visible at the bottom of the tube, and some protein precipitate may also float on the liquid surface.

9.Carefully pipette or pour the supernatant (approximately 450 µL) into a new 1.5 mL centrifuge tube. Avoid aspirating the protein pellet at the bottom or floating on the surface. If protein precipitate is accidentally transferred to the new tube, centrifuge again for 2 minutes and collect the supernatant.

10.Add an equal volume (300-450 µL) of room temperature isopropanol, gently invert 30 times to mix until flocculent (filamentous) white DNA precipitate appears.
Note: Sometimes the flocculent (filamentous) DNA precipitate adheres to the lid or tube mouth during inversion and cannot be dislodged, or floats on the liquid surface due to attached bubbles. This may cause the operator to fail to see the precipitate and mistakenly assume no DNA was obtained.

11.Centrifuge at 12,000 rpm for 1 minute. A white DNA pellet will be visible at the bottom of the tube. Discard the supernatant.

12.Add 500 µL of 70% ethanol, invert several times to wash the DNA pellet, then centrifuge at 12,000 rpm for 1 minute. Discard the supernatant (be careful not to discard the DNA pellet if it adheres loosely to the tube wall). Check for the presence of the white DNA pellet. Use a yellow pipette tip to transfer any DNA adhering to the lid or tube mouth to the bottom of the centrifuge tube. Invert all EP tubes on absorbent paper and tap gently to drain residual ethanol, or carefully aspirate residual ethanol around the pellet and tube wall with a pipette tip. Air-dry the pellet for a few minutes. Avoid over-drying (DNA will be extremely difficult to dissolve) or leaving excessive residual ethanol (which may inhibit downstream reactions such as enzyme digestion).

13.Add 100 µL of DNA Dissolution Buffer to redissolve the DNA pellet. Tap the tube wall to mix. Incubate at 65℃ for 30-60 minutes (no more than 1 hour), tapping the tube wall occasionally to assist hydration. Alternatively, allow hydration at room temperature or 4℃ for 1-5 days.

14.Store DNA at 2-8℃ for short-term use or -20℃ for long-term storage.