Animal/Cell Genomic DNA Mini Extraction Kit

1. Components of the reagent kit

2. Storage

This reagent kit should be stored at room temperature (15-25℃) and in dry conditions, with a shelf life of 12 months. The DNA extraction purification columns can be stored for 1 year in a cool and dry environment. Proteinase K and RNase A contain preservatives, allowing transportation at room temperature, but for long-term storage, they should be kept at -20℃.

3. Instructions for Using the Reagent Kit

3.1 This reagent kit is intended for molecular biology research and should not be used for disease diagnosis or treatment.

3.2 Some components in the reagent kit contain irritants. Protective measures such as wearing protective clothing and goggles are recommended.

3.3 During the usage of this reagent kit, a high-speed centrifuge, water bath (metal bath), vortex mixer, anhydrous ethanol, sterile deionized water, and EP tubes need to be prepared by the user.

4. Introduction to the Reagent Kit

The Animal/Tissue DNA Purification Kit offers a rapid and effective solution for animal tissue and cell culture DNA purification, widely applicable across animal tissues and cell cultures.

This DNA rapid purification kit swiftly extracts total DNA from animals and cells, yielding DNA directly usable for PCR, Southern blotting, and similar applications. The purification process does not require toxic agents like phenol-chloroform, making this DNA purification kit highly suitable for various other samples.

5. Experimental Principles and Procedures

6. Extraction Process

Before Starting the Experiment:

A. Reagent Buffer IV tends to precipitate under low-temperature conditions. It is recommended to heat at 65℃ for 5 minutes. After the precipitation dissolves, it can be used normally.

B. Prior to use, add the specified amount of anhydrous ethanol to WashBuffer 1 as indicated on the reagent bottle label, and mark a check on the label to indicate the addition of anhydrous ethanol.

C. Elution Buffer is a 0.1x TE solution with minimal EDTA content. If EDTA might affect subsequent experiments, it is recommended to replace the Elution Buffer with sterile deionized water.

1. Sample Handling:

A. Take cell culture, centrifuge at 12,000 rpm for 1 minute to collect cells, and aspirate the supernatant as much as possible. Add 250μl of Reagent Buffer IV and 10μl of RNaseA (10 mg/ml) to the collected cells, thoroughly suspend, and incubate at 65°C for 10 minutes.

B. Grind tissue not exceeding 25 mg into a fine powder using liquid nitrogen. Add 200μl of Reagent Buffer IV, 20μl of Proteinase K (10 mg/ml), and 10μl of RNaseA (10 mg/ml) to the powder, shake well, and incubate at 65°C for 30 minutes, shaking the mixture four times during this period.

2. Add 200μl of Reagent Buffer Cplus to the lysate and mix well. If a white precipitate appears, it can be left undisturbed; it won’t affect subsequent experiments once the precipitate disappears.

3. Add 200μl of anhydrous ethanol and mix thoroughly. Some precipitation might occur, but it won’t affect subsequent experiments.

4. Apply the obtained liquid to the DNA extraction purification column (sleeve) (approximately 650~700μl each time), let it sit at room temperature for 2 minutes, centrifuge at 12,000 rpm for 30 seconds, discard the collected waste, and re-insert the collection tube into the purification column for the next step.

5. Place the DNA extraction purification column (sleeve) into a new collection sleeve, add 700μl of WashBuffer 1, centrifuge at 12,000 rpm for 30 seconds, discard the waste, and place the DNA extraction purification column (sleeve) back into the sleeve for the next step.

(Note: Confirm the addition of anhydrous ethanol to Rinse Buffer 1.)

6. Add 500μl of WashBuffer 1 to the DNA extraction purification column (sleeve), centrifuge at 12,000 rpm for 30 seconds, extend centrifugation time appropriately to ensure the membrane is drier.

(Note: Ethanol has a significant impact on subsequent experiments; therefore, membrane dryness is crucial. After centrifugation, ensure no ethanol remains before elution. Discard the waste and collection tubes afterward. After rinsing with Wash Buffer 1, the membrane on the DNA purification column should have a faint color. Carefully remove the DNA purification column after centrifugation, ensuring it doesn’t touch the collection tube to prevent ethanol contamination.)

7. Place the DNA purification column in a new centrifuge tube, add 100μl of Elution Buffer directly onto the membrane, centrifuge at 12,000 rpm for 1 minute, and collect the DNA.

(Note: Eluting DNA with 50μl of Elution Buffer can increase DNA concentration but decreases total DNA yield.)