Products

Micro/Clinical gDNA Extraction Kit(SpinColumn)

Packing Specification：

Product Introduction：
This kit adopts special imported DNA adsorption columns and a unique buffer system, specially designed for the isolation and purification of genomic DNA from micro-samples such as micro-volume blood, forensic materials, dried blood spots, swabs, chewing gum, and urine. After lysis and digestion of samples from various sources, DNA is selectively adsorbed onto the silica membrane of the spin column under high chaotropic salt conditions (Acryl Carrier is specially provided to easily capture trace nucleic acids from the system). Through a series of rapid washing and centrifugation steps, impurities such as salts, cellular metabolites, and proteins are removed. Finally, pure genomic DNA is eluted from the silica membrane using a low-salt Elution Buffer. The purified DNA is free of impurities and PCR inhibitors, and can be directly used for PCR analysis.

Product Features：
1.No need for toxic reagents such as phenol or ethanol precipitation steps.
2.Time-saving and simple; operation for a single sample can generally be completed within 20 minutes.
3.Equipped with Acryl Carrier for full collection of extremely trace DNA.
4.Multiple column washes ensure high purity. The extracted DNA has high purity and stable quality, suitable for various routine operations including PCR, enzyme digestion, sequencing, and Southern hybridization.

Application Scope：
Suitable for the isolation and purification of genomic DNA from micro-samples such as micro-volume blood, forensic materials, dried blood spots, and swabs.

Operation Steps：
Tip: 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!
1.Blood Samples
a.Take 1-100 μL of blood into a 1.5 mL centrifuge tube.
b.If the volume is less than 100 μL, make up to 100 μL with Lysis Buffer ML.
c.Add 10 μL of Proteinase K (20 mg/mL) solution, mix thoroughly. Add 100 μL of Binding Buffer CB, immediately vortex to mix well, and incubate at 70℃ for 10 minutes.
For processing < 10 μL of sample, it is recommended to add 1 μL of Acryl Carrier stock solution to 100 μL of Binding Buffer CB.
d.After cooling, add 50 μL of anhydrous ethanol, immediately vortex to mix thoroughly, and incubate at room temperature for 3 minutes.
If the ambient temperature exceeds 25℃, pre-cool the anhydrous ethanol on ice before adding.
e.Proceed to Step 7 under "Common Operation Steps".

2.Dried Blood Spots
a.Punch 3 mm (1/8 inch) diameter pieces from the blood card (with dried blood spots) using a puncher. Place up to 3 such pieces into a 1.5 mL centrifuge tube.
Blood should generally be spotted and dried on specific paper or blood cards, such as 903 paper or IsoCode paper (Schleicher & Schuell), Bloodstain Card or FTA Card (Whatman), Guthrie test cards, or comparable blood cards.
b.Add 180 μL of Lysis Buffer ML.
c.Add 20 μL of Proteinase K (20 mg/mL) solution, immediately vortex to mix thoroughly.
d.Shake at 900 rpm on a 56℃ orbital shaker for 1 hour.
If a heated orbital shaker is not available, use a water bath or heating block, vortexing for 10 seconds every 10 minutes to assist lysis.
e.Add 200 μL of Binding Buffer CB, immediately vortex to mix thoroughly.
For processing only one 3 mm blood card piece, it is recommended to add 2 μL of Acryl Carrier stock solution to 200 μL of Binding Buffer CB.
f.Shake at 900 rpm on a 70℃ orbital shaker for 10 minutes.
If a heated orbital shaker is not available, use a water bath or heating block, vortexing for 10 seconds every 3 minutes to assist lysis.
g.Proceed to Step 7 under "Common Operation Steps".

3.Tissue Samples
a.Grind fresh or thawed tissue into fine powder in liquid nitrogen or cut into small pieces with a scalpel (cutting into small pieces can improve yield). Take < 10 mg and transfer into a 1.5 mL centrifuge tube containing 180 μL of Lysis Buffer ML. Pipette to mix with a large-bore pipette tip.
b.Add 20 μL of Proteinase K (20 mg/mL) solution, immediately vortex to mix thoroughly.
c.Incubate the lysate in a 56℃ water bath overnight or until complete tissue digestion, gently shaking several times during incubation to assist lysis.
For small amounts of tissue, digestion usually takes 4-6 hours, but overnight digestion yields the best results.
d.Add 200 μL of Binding Buffer CB, immediately vortex to mix thoroughly.
For processing small sample amounts, it is recommended to add 2 μL of Acryl Carrier stock solution to 200 μL of Binding Buffer CB.
e.After cooling, add 200 μL of anhydrous ethanol, immediately vortex to mix thoroughly, and incubate at room temperature for 5 minutes.
If the ambient temperature exceeds 25℃, pre-cool the anhydrous ethanol on ice before adding.
f.Proceed to Step 7 under "Common Operation Steps".

4.Chewing Gum
a.Cut 30 mg of chewing gum into small pieces and place into a 1.5 mL centrifuge tube. Add 280 μL of Lysis Buffer ML and 20 μL of Proteinase K (20 mg/mL) solution, immediately vortex to mix thoroughly.
b.Shake at 900 rpm on a 56℃ orbital shaker for at least 3 hours.
If a heated orbital shaker is not available, use a water bath or heating block, vortexing for 10 seconds every 10 minutes to assist lysis.
c.Add 200 μL of Binding Buffer CB (with 2 μL of Acryl Carrier), immediately vortex to mix thoroughly.
d.Shake at 900 rpm on a 70℃ orbital shaker for 1 hour.
If a heated orbital shaker is not available, use a water bath or heating block, vortexing for 10 seconds every 10 minutes to assist lysis.
e.After cooling, add 200 μL of anhydrous ethanol, immediately vortex to mix thoroughly.
f.Centrifuge at maximum speed (approximately 14,000 rpm) for 1 minute. Load the supernatant into a Spin AC column (placed in a collection tube), centrifuge at 13,000 rpm for 30-60 seconds, and discard the waste liquid in the collection tube.
g.Proceed to Step 8 under "Common Operation Steps".

5.Forensic Materials
a1.Cigarette butt/filter paper: Cut 1 cm² of cigarette butt or outer filter paper into 6 small pieces, place into a 1.5 mL centrifuge tube. Add 300 μL of Lysis Buffer ML and 20 μL of Proteinase K (20 mg/mL) solution, immediately vortex to mix thoroughly. Proceed to Step b.
a2.Envelope/stamp: Cut 0.5-2.5 cm² of envelope or stamp into small pieces, place into a 1.5 mL centrifuge tube. Add 300 μL of Lysis Buffer ML and 20 μL of Proteinase K (20 mg/mL) solution, immediately vortex to mix thoroughly. Proceed to Step b.
a3.Hair: Cut 0.5-1 cm of hair from the root follicle, place into a 1.5 mL centrifuge tube. Add 280 μL of Lysis Buffer ML, 20 μL of Proteinase K (20 mg/mL) solution, and 20 μL of 1 M DTT solution, immediately vortex to mix thoroughly. Proceed to Step b.
a4.Nail: Cut nails into small pieces, place into a 1.5 mL centrifuge tube. Add 280 μL of Lysis Buffer ML, 20 μL of Proteinase K (20 mg/mL) solution, and 20 μL of 1 M DTT solution, immediately vortex to mix thoroughly. Proceed to Step b.
a5.Blood/saliva/semen-stained envelope: Cut approximately 0.5 cm² of the stained material into small pieces, place into a 1.5 mL centrifuge tube. Add 300 μL of Lysis Buffer ML and 20 μL of Proteinase K (20 mg/mL) solution (add an additional 20 μL of 1 M DTT solution for semen), immediately vortex to mix thoroughly. Proceed to Step b.
a6.Shake at 900 rpm on a 56℃ orbital shaker for 1 hour.
If a heated orbital shaker is not available, use a water bath or heating block, vortexing for 10 seconds every 10 minutes to assist lysis. For hair, lysis is usually complete in 1 hour (extend if incomplete). For hard-to-lyse samples such as nails, overnight lysis is recommended. Undigested insoluble materials will be removed by centrifugation in subsequent Step e.
b.Add 200 μL of Binding Buffer CB (with 2 μL of Acryl Carrier), immediately vortex to mix thoroughly.
c.Shake at 900 rpm on a 70℃ orbital shaker for 1 hour.
d.Centrifuge at maximum speed (approximately 14,000 rpm) for 1 minute. Load the supernatant into a Spin AC column (placed in a collection tube), centrifuge at 13,000 rpm for 30-60 seconds, and discard the waste liquid in the collection tube.
e.Proceed to Step 8 under "Common Operation Steps".

6.Microdissected Samples (Including Formalin-Fixed Microdissected Samples)
a.Add 15 μL of Lysis Buffer ML to a 0.2 mL centrifuge tube and place the microdissected sample into it.
b.Add 10 μL of Proteinase K (20 mg/mL) solution, immediately vortex to mix thoroughly.
c.Incubate in a 56℃ water bath for 3 hours (16 hours for formalin-fixed samples) until complete lysis, inverting and vortexing occasionally.
d.Add 15 μL of Lysis Buffer ML, then add 50 μL of Binding Buffer CB (with 1 μL of Acryl Carrier), immediately vortex to mix thoroughly.
e.After cooling, add 50 μL of anhydrous ethanol, immediately vortex to mix thoroughly, and incubate at room temperature for 5 minutes.
If the ambient temperature exceeds 25℃, pre-cool the anhydrous ethanol on ice before adding.
f.Proceed to Step 7 under "Common Operation Steps".

7.Load the mixture (including any precipitation) into a Spin AC column (placed in a collection tube). Centrifuge at 13,000 rpm for 30-60 seconds and discard the waste liquid in the collection tube.

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

9.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.

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

11.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).

12.Remove the Spin AC column and place it into a clean centrifuge tube. Add 20-30 μL of Elution Buffer EB (preheating the elution buffer in a 65-70℃ water bath beforehand improves elution efficiency) to the center of the adsorption membrane. Incubate at room temperature for 2-5 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.
Larger elution volumes result in higher elution efficiency. For higher DNA concentration, reduce the elution volume (minimum 20 μL; volumes smaller than this reduce elution efficiency and DNA yield).

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