Products

0.1-1mL Coagulated Blood gDNA Extraction Kit(SpinColumn)

Packing Specification：

Product Introduction:
This kit enables efficient extraction of genomic DNA from coagulated blood clots. The unique combination of Binding Buffer and Proteinase K rapidly lyses cells and inactivates intracellular nucleases. Genomic DNA is then selectively adsorbed onto the silica membrane of the spin column under high chaotropic salt conditions. Through a series of rapid washing and centrifugation steps, Inhibitor Removal Buffer and Wash Buffer remove impurities such as cellular metabolites and proteins. Finally, pure genomic DNA is eluted from the silica membrane using a low-salt Elution Buffer.

Product Features:
1.No need for toxic reagents such as phenol or ethanol precipitation steps.
2.Fast and simple; operation for a single sample can generally be completed within 90 minutes.
3.Multiple column washes ensure high purity. Typically, 3-6 µg of DNA can be extracted from 200 µL of whole blood, with a typical OD260/OD280 ratio of 1.7-1.9 and a length of 30 kb - 50 kb. The DNA can be directly used for PCR, Southern blot, and various restriction enzyme digestion reactions.

Application Scope:
Suitable for the rapid extraction of genomic DNA from 0.1-1 mL (1-2 mL as described) coagulated blood clots.

Operation Steps：
Tips:
Before first use, add the specified amount of anhydrous ethanol to Wash Buffer WB, mix thoroughly, and mark the box to indicate ethanol has been added to avoid repeated addition!
10×Red Blood Cell Lysis Buffer must be diluted to 1× working solution before use; store in a self-prepared blue-mouth bottle.
Pretreat the spin column with Equilibration Buffer (mandatory step; see "Use of Equilibration Buffer" above for detailed method).

1.Take approximately 0.1-1 mL (about 100-300 mg) of the sample containing coagulated blood clots. Place the clots into a mortar and grind into powder with liquid nitrogen. Alternatively, transfer the clots into a round-bottom centrifuge tube, add 3-5 grinding zirconium beads (4-5 mm in diameter), and homogenize the clots by high-frequency oscillation in a tissue grinder for 1-2 minutes. Transfer the homogenized filtrate or liquid nitrogen-ground coagulated blood powder into a pre-prepared 15 mL large-volume centrifuge tube (self-prepared) in batches.

2.Add 6 mL of 1×Red Blood Cell Lysis Buffer to the 15 mL centrifuge tube, invert 6-8 times, vortex to mix well, and incubate at room temperature for 10 minutes. Invert and tap the tube several times during incubation to facilitate red blood cell lysis.

3.Centrifuge at the maximum speed of 4,000 rpm with a desktop centrifuge for 3-5 minutes. Aspirate and discard the red supernatant, leaving an intact white blood cell pellet at the bottom of the tube with approximately 10 μL of residual supernatant. If large clots remain, repeat Steps 1-2.

4.Transfer the white blood cell pellet into a 1.5 mL centrifuge tube containing 360 μL of Tissue Lysis Buffer TL, and immediately vortex to resuspend the pellet.

5.Add 40 μL of Proteinase K solution (20 mg/mL) and immediately vortex to mix thoroughly.

6.Incubate the lysate in a 65℃ water bath for 45-60 minutes or until complete tissue digestion, gently shaking several times during incubation to assist lysis. If undissolved clots persist, cool the sample first, then repeatedly aspirate 10 times with a 1 mL disposable syringe without a needle (self-prepared) or a 1-2 mL disposable plastic Pasteur pipette (self-prepared), and continue incubation in the 65℃ water bath until complete digestion.
Optional step: If significant RNA contamination is present, add 40 μL of RNase A (25 mg/mL, self-prepared) solution after completing Step 6, vortex to mix, and incubate at room temperature for 5-10 minutes.

7.Add 400 μL of Binding Buffer CB, immediately vortex to mix thoroughly, and incubate at 70℃ for 15 minutes.

8.After cooling, add 200 μL of isopropanol and immediately vortex to mix thoroughly (flocculent precipitation may occur).

9.Use a 1 mL pipette tip to aspirate the mixture, and load approximately 1000 μL of the mixture into a Spin AC column (placed in a collection tube) in 2 batches. Centrifuge at 13,000 rpm for 60 seconds and discard the waste liquid in the collection tube.
Note: Complete dissolution of the coagulated blood clots and immediate vortexing or pipetting to mix thoroughly are critical in this step. Inadequate mixing will severely reduce yield. For viscous samples that are difficult to mix, vortex for 15 seconds if necessary.

10.Add 500 μL of Inhibitor Removal Buffer IR, centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid.

11.Add 600 μL of Wash Buffer WB (ensure anhydrous ethanol has been added!), centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid.

12.Add another 600 μL of Wash Buffer WB, centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid.

13.Place the Spin AC column back into the empty collection tube and centrifuge at 13,000 rpm for 2 minutes to thoroughly remove residual Wash Buffer (residual ethanol may inhibit downstream reactions).

14.Remove the Spin AC column and place it into a clean centrifuge tube. Add 100 μL of Elution Buffer EB (preheating the elution buffer in a 65-70℃ water bath beforehand) to the center of the adsorption membrane. Incubate at room temperature for 3-5 minutes, then centrifuge at 12,000 rpm for 1 minute. Transfer the eluted solution back into the spin column, incubate at room temperature for 2 minutes, and centrifuge at 12,000 rpm for 1 minute.
Note: Larger elution volumes result in higher elution efficiency. For higher DNA concentration, reduce the elution volume (minimum 50 μL; volumes smaller than this reduce elution efficiency and DNA yield).

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