Products

Blood gDNA Isolation Midi 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.

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 60 minutes.
4.Stable results and high yield: typically, 50-150 µg of DNA can be extracted from 3 mL 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.

Application Scope:
Suitable for the rapid extraction of genomic DNA from whole blood of various animals.

Operation Steps:
This kit is solution-based and can process blood volumes scaled up or down in proportion (20 µL-10 mL)!
1.Pipette 7-9 mL of 1x Red Blood Cell Lysis Buffer (dilute 10× stock to 1x with deionized water before use) into a 15 mL centrifuge tube.

2.After bringing anticoagulated whole blood to room temperature and inverting to mix, pipette 3 mL of whole blood into the 15 mL 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, inverting to mix 6-8 times during incubation. Alternatively, use a vortex mixer or shaker to facilitate red blood cell lysis.

4.Centrifuge at 2,500 x g (4,000 rpm) with a desktop centrifuge for 2 minutes. Discard the red supernatant and invert the centrifuge tube on absorbent paper to remove residual blood from the tube mouth.
Optional step: Add another 1-3 mL of Red Blood Cell Lysis Buffer, invert several times, incubate for 1-2 minutes, centrifuge at 2,500 x g (4,000 rpm) for 2 minutes, then discard the red supernatant. Two rounds of red blood cell lysis result in higher DNA purity.

5.Add 3 mL of Nucleus Lysis Buffer and use a Pasteur pipette to pipette up and down rapidly and vigorously 10-15 times to resuspend and disperse the white blood cell pellet (this step is critical and cannot be omitted). Then incubate at a constant temperature of 60-65℃ in a water bath or oven for approximately 30-45 minutes, depending on the lysis status of white blood cells.
Note: Vigorous vortexing and rapid pipetting can help the Nucleus Lysis Buffer contact and lyse white blood cells. As genomic DNA is released immediately, the mixture will become very viscous—stop pipetting at this point to avoid shearing genomic DNA.

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 1 mL of Protein Precipitation Buffer, and vortex continuously at high speed for 25 seconds. Small protein clumps should be visible after mixing. Alternatively, use a Pasteur pipette to pipette up and down rapidly and vigorously 5-10 times for better mixing.

8.Centrifuge at 2,500 x g (4,000 rpm) for 5-10 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 4 mL) into a new 15 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 (3-4 mL) of room temperature isopropanol, gently invert 30 times to mix until flocculent (filamentous) white DNA precipitate appears.

Note: After DNA transfer, wipe the marked number on the 15 mL centrifuge tube with isopropanol, discard the solution in the tube, and soak the tube in a plastic bucket containing disinfectant tablets or laundry detergent for half a day. Rinse the centrifuge tube thoroughly with tap water, soak in 84 disinfectant for one day, rinse repeatedly with clean water, and finally autoclave for reuse.

11.Use a yellow pipette tip to transfer the DNA precipitate to a clean 1.5 mL EP tube and aspirate the supernatant completely. Add 300-500 µL of 70% ethanol, invert several times to wash the DNA precipitate, centrifuge at 12,000 rpm for 1 minute, and discard the supernatant (be careful not to discard the DNA precipitate). Tap gently on absorbent paper 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).

12.Add 100-500 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℃ overnight.
Store DNA at 2-8℃ for short-term use or -20℃ for long-term storage.