Products

Tissue/Cell gDNA Isolation Kit(Solution)

Packing Specification：

Product Introduction:
This kit is used for the rapid extraction of genomic DNA from animal and plant cells/tissues. After grinding or homogenizing the sample, add Nucleus Lysis Buffer. First, cells are lysed to release genomic DNA under the synergistic effect of strong detergents and Proteinase K (if used). Then RNase A is added to remove RNA, followed by selective precipitation of proteins with Protein Precipitation Buffer. Finally, pure genomic DNA is precipitated with isopropanol and redissolved in DNA Dissolution Buffer.

Product Features:
1.No need for toxic reagents such as phenol or chloroform.
2.Fast and simple; the entire process for tissue samples can be completed within 1 hour.
3.Stable results and high yield (more than twice the yield of spin column-based kits). The typical OD260/OD280 ratio is 1.7-1.9, and the DNA length can reach 50 kb-150 kb. It can be directly used for PCR, Southern blot, various restriction enzyme digestion reactions, and library construction.

Application Scope:
Suitable for the rapid extraction of genomic DNA from various animal and plant cells/tissues.

Operation Steps:
1.Cultured Tissue Cells
a.Collect cells into a 1.5 mL centrifuge tube. For adherent cells, digest with trypsin first, then pipette to detach and collect.
b.Centrifuge at 13,000 rpm for 10 seconds to pellet the cells. Discard the supernatant, leaving the cell pellet with approximately 10-50 μL of residual liquid.
c.Add 200 μL of PBS to resuspend and wash the cells. Repeat the previous centrifugation step, then vortex vigorously to resuspend the cell pellet.
d.For cell lines with poor lysis efficiency using Nucleus Lysis Buffer (e.g., PC12 cells), perform several freeze-thaw cycles before the next step: freeze in liquid nitrogen, thaw in a 95℃ water bath, and repeat 4 times.
e.Add 600 μL of Nucleus Lysis Buffer. Gently pipette with a large-bore pipette tip (cut off the tip) to lyse the cells until no cell clumps are visible.
f.Proceed to Step 4 under "Common Operation Steps".

2.Animal and Plant Tissues (e.g., Mouse Liver, Brain, or Plant Leaves)
a.Add 600 μL of ice-precooled Nucleus Lysis Buffer to 10-20 mg of fresh or thawed tissue. Homogenize with a small homogenizer for 10 seconds, then transfer the lysate to a 1.5 mL centrifuge tube. Alternatively: Grind 10-20 mg of tissue (40 mg for plant leaves) into fine powder in liquid nitrogen, transfer to a 1.5 mL centrifuge tube containing 600 μL of ice-precooled Nucleus Lysis Buffer, and pipette to mix with a large-bore pipette tip.
b.Incubate the lysate in a 65℃ water bath for 15-30 minutes.
c.Proceed to Step 4 under "Common Operation Steps".

3.Animal Tissue (Mouse Tail)
a.Before processing the sample, add 120 μL of 0.5 M EDTA (pH 8.0) to a 1.5 mL centrifuge tube containing 500 μL of Nucleus Lysis Buffer, mix well, and cool on ice for later use.
b.Grind the mouse tail into fine powder in liquid nitrogen, or cut 0.5-1.0 cm of the mouse tail tip (ensure to cut within 0-2 cm of the tail tip for optimal lysis) into small pieces and place into a 1.5 mL centrifuge tube. Add 600 μL of the EDTA/Nucleus Lysis Buffer prepared in the previous step.
c.Add 17.5 μL of Proteinase K solution (20 mg/mL) and invert to mix.
d.Incubate overnight in a 55℃ water bath, gently shaking several times during incubation to assist lysis. Alternatively, incubate in a 55℃ water bath on a shaker for 3 hours, vortexing vigorously once every hour. Ensure complete lysis of the mouse tail (undercut mouse tails may not lyse completely, resulting in lower yield).

4.Add 1.8 μL of RNase A (10 mg/mL) to the lysate to a final concentration of 30 μg/mL. Invert to mix, then incubate at 37℃ for 15-30 minutes to remove residual RNA. Cool to room temperature for at least 5 minutes or on ice.

5.Add 200 μL of Protein Precipitation Buffer to the room temperature lysate, then vortex continuously at high speed for 25 seconds. Small protein clumps may be visible after mixing. Incubate on ice for 5 minutes.
Note: Due to the small sample volume and weight, the shearing force generated by vortex mixing will not shear genomic DNA. If mixing by hand, do not shake vigorously up and down—only mix with moderate force to avoid shearing DNA. However, the force must be sufficient to ensure thorough mixing and dispersion of the viscous lysate; otherwise, DNA cannot be separated from protein precipitates and will precipitate with proteins during centrifugation, causing DNA loss or reduced yield. Inadequate mixing may also result in incomplete protein precipitation, leading to significant protein contamination in the final product. Therefore, vortex mixing is recommended.

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

7.Carefully and slowly aspirate the supernatant into a new 1.5 mL centrifuge tube, avoiding the precipitate.
Do not aspirate 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.

8.Add an equal volume (approximately 600 μL) of room temperature isopropanol, invert 30 times to mix until flocculent (filamentous) white DNA precipitate appears.
During inversion mixing, flocculent (filamentous) DNA may sometimes adhere to the lid or tube mouth and not dislodge, leading the operator to fail to see the precipitate and mistakenly assume no DNA was obtained. Solution: Omit Step 9, centrifuge directly at 12,000 rpm for 1 minute, discard the supernatant, then proceed to Step 11.

9.Place the centrifuge tube vertically to allow the white DNA precipitate to settle naturally to the bottom. Aspirate and discard as much supernatant as possible, taking care not to aspirate the precipitate.
If the flocculent (filamentous) DNA precipitate has attached bubbles, it will float on the liquid surface instead of settling. Carefully aspirate the supernatant while avoiding the precipitate.

10.Add 1 mL of 70% ethanol, invert to wash the DNA precipitate, then centrifuge at 12,000 rpm for 1 minute. A white DNA pellet will be visible at the bottom of the tube. Discard the supernatant.

11.Add another 1 mL of 70% ethanol, invert several times to wash the DNA precipitate, then centrifuge at 12,000 rpm for 1 minute. Discard the supernatant (be careful not to discard the DNA pellet). Invert the tube and 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).
Add 100 μL of DNA Dissolution Buffer to rehydrate and dissolve 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.