Products

Tissue/Cell gDNA Extraction Kit(96 well plate)

Packing Specification：

Product Introduction:
A unique binding buffer/Proteinase K rapidly lyses cells and inactivates intracellular nucleases. Genomic DNA is then selectively adsorbed onto the silica matrix membrane in the spin column under high chaotropic salt conditions. Impurities such as cellular metabolites and proteins are removed by the inhibitor removal buffer and washing buffer through a series of rapid washing-centrifugation steps. Finally, pure genomic DNA is eluted from the silica matrix membrane using a low-salt elution buffer.

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

Product Features:
1.The silica matrix membranes in the 96-well adsorption plate are all high-quality special adsorption membranes, with minimal variation in adsorption capacity between columns.
2.No toxic reagents such as phenol are required, nor are steps like ethanol precipitation.
3.Fast and simple: operation for a single sample can usually be completed within 30 minutes.
Multiple column washing steps ensure high purity. The typical OD260/OD280 ratio ranges from 1.7 to 1.9, with a length of up to 30 kb-50 kb. The DNA can be directly used for PCR, Southern-blot, and various restriction enzyme digestion reactions.

Operation Steps: 
Pre-Experiment Preparation:
1.Before first use, add the specified amount of absolute ethanol to Washing Buffer WB, mix thoroughly. After addition, mark the corresponding box to indicate ethanol has been added to avoid repeated additions!
2.Pre-treat the adsorption column with equilibration buffer for later use: Pre-treatment of the silica gel membrane adsorption column with equilibration buffer is a mandatory step; refer to "Regarding the Use of Equilibration Buffer" above for specific methods.

Operation Steps (Centrifugation Method)
1.Cultured Cells:
a.Collect approximately 105-106 suspension cells into a 1.5 mL centrifuge tube; for adherent cells, digest with trypsin first, then pipette to collect.
b.Centrifuge at 13,000 rpm for 10 seconds to pellet the cells. Discard the supernatant, leaving the cell pellet and approximately 10-20 μL of residual liquid.
c.Add 200 μL of 1× PBS to resuspend and wash the cells, centrifuge at 13,000 rpm for 10 seconds to pellet the cells. Completely aspirate and discard the supernatant, and resuspend the cell pellet in 180 μL of 1× PBS.
d.Add 20 μL of Proteinase K (20mg/mL) solution, mix thoroughly, then add 200 μL of Binding Buffer CB, immediately vortex vigorously to mix well, incubate at 70°C for 10 minutes, cool to room temperature, and set aside.
Optional step: If RNA contamination is significant and needs to be removed, add 4 μL of RNase A (10mg/mL) solution before adding 200 μL of Binding Buffer CB, vortex to mix, and incubate at room temperature for 5-10 minutes.
e.Proceed to Step 3 under the General Extraction Steps section.

2.Animal and Plant Tissues (e.g., Mouse Liver/Brain or Mouse Tail):
a.Take 20-50mg of fresh or thawed tissue, grind it into a fine powder in liquid nitrogen, transfer to a 1.5 mL centrifuge tube containing 180 μL of Tissue Lysis Buffer TL, and pipette to mix with a large-bore tip.
b.Add 20 μL of Proteinase K solution (20mg/mL), immediately vortex vigorously to mix well.
c.Incubate the lysate in a 55℃ water bath for 1-3 hours or until complete tissue digestion, gently vortex several times during incubation to assist lysis.
Optional step: If RNA contamination is significant and needs to be removed, add 4 μL of RNase A (10mg/mL) solution after completing Step c, vortex to mix, and incubate at room temperature for 5-10 minutes.
d.Add 200 μL of Binding Buffer CB, immediately vortex vigorously to mix well, incubate at 70°C for 10 minutes. If there are undigested tissue/cell samples, centrifuge at 12,000 rpm for 1 minute and collect the supernatant for use.
e.Proceed to Step 3 under the General Extraction Steps section.

3.Add 100 μL of isopropanol to the mixture from the previous step, pipette 10-20 times to mix thoroughly. Flocculent precipitation may occur at this time.
Tips: Thorough mixing by pipetting is very important; insufficient mixing will seriously reduce yield.

4.Transfer the mixture from the previous step to the 96-well Silicon Plate (SP), place the 96-well SP on a 96-well Deep Well Plate, and cover with a sealing film. Centrifuge at 5,000 rpm for 5 minutes, discard the waste liquid in the Deep Well Plate, and place the 96-well SP back on the Deep Well Plate.

5.Add 500 μL of Inhibitor Removal Buffer IR to the 96-well SP, cover with a sealing film, centrifuge at 5,000 rpm for 5 minutes, discard the waste liquid in the Deep Well Plate, and place the 96-well SP back on the Deep Well Plate.

6.Add 600 μL of Washing Buffer WB (check if absolute ethanol has been added before use!) to the 96-well SP, cover with a sealing film. Centrifuge at 5,000 rpm for 2 minutes, discard the waste liquid in the Deep Well Plate, and place the 96-well SP back on the Deep Well Plate.

7.Repeat Step 4 once: Add another 600 μL of Washing Buffer WB to the 96-well SP, cover with a sealing film. Centrifuge at 5,000 rpm for 5 minutes, discard the waste liquid in the Deep Well Plate, and place the 96-well SP back on the Deep Well Plate.

8.Place the 96-well SP back in the 96-well Deep Well Plate, centrifuge at 5,000 rpm for 5 minutes, and discard the waste liquid. Air-dry the adsorption membrane at room temperature for 3-5 minutes to completely remove residual Washing Buffer, as residual ethanol in the Washing Buffer may inhibit downstream reactions.

9.Transfer the 96-well SP to a clean 96-well Deep Well Plate. Add 60-150 μL of Elution Buffer EB (pre-warming the elution buffer in a 65-70℃ water bath beforehand improves elution efficiency) dropwise to the center of the adsorption membrane without touching it. Incubate at room temperature for 3-5 minutes, centrifuge at 3,600 rpm (~2,130 x g) for 8 minutes to collect the DNA solution into the 96-well Deep Well Plate. To increase the yield of genomic DNA, transfer the eluted DNA solution back into the 96-well SP, place the 96-well SP on the Deep Well Plate, centrifuge at 3,600 rpm (~2,130 x g) for 8 minutes to collect the DNA solution into the 96-well Deep Well Plate. The DNA can be transferred to a PCR plate using a multi-channel pipette and sealed with a sealing film.
Larger elution volumes result in higher elution efficiency. If higher DNA concentration is required, the elution volume can be appropriately reduced, but the minimum volume should not be less than 50 μL (excessively small volumes reduce elution efficiency and DNA yield).

10.DNA can be stored at 2-8℃ for short-term use, or at -20℃ for long-term storage.

Operation Steps (Vacuum Negative Pressure Method)

If using the vacuum negative pressure method for extraction, a vacuum negative pressure device must be prepared by the user. Steps 1 and 2 (sample pre-treatment) are the same as the centrifugation method.
1.Place the waste liquid tank under the negative pressure device, place the 96-well SP on the negative pressure device, transfer the supernatant of the mixture from the previous step to the 96-well SP, turn on the negative pressure device, and vacuum filter for 5 minutes.

2.Add 500 μL of Inhibitor Removal Buffer IR to the 96-well SP, turn on the negative pressure device, and vacuum filter for 2 minutes.

3.Add 600 μL of Washing Buffer WB (check if absolute ethanol has been added before use!) to the 96-well SP, turn on the negative pressure device, and vacuum filter for 2 minutes.

4.Repeat Step 3 once: Add another 600 μL of Washing Buffer WB to the 96-well SP, turn on the negative pressure device, and vacuum filter for 5 minutes.

5.Turn off the vacuum negative pressure device, clean the waste liquid tank, and air-dry the 96-well SP at room temperature for 3 minutes.

6.Place the 96-well SP on the negative pressure device with a new 96-well Deep Well Plate underneath. Add 60-150 μL of Elution Buffer EB (pre-warming the elution buffer in a 65-70℃ water bath beforehand improves elution efficiency) dropwise to the center of the adsorption membrane without touching it. Incubate at room temperature for 3-5 minutes, turn on the negative pressure device, and filter for 3-5 minutes. To increase the yield of genomic DNA, transfer the eluted DNA solution back into the 96-well SP, turn on the negative pressure device, and filter for 3-5 minutes. Collect the DNA solution into the 96-well Deep Well Plate by centrifugation. The DNA can be transferred to a PCR plate using a multi-channel pipette and sealed with a sealing film.

7.DNA can be stored at 2-8℃ for short-term use, or at -20℃ for long-term storage.