Bacterial Genome DNA Extraction Kit

• Components of the reagent kit

• ストレージ

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

• 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, 水浴 (metal bath), vortex mixer, anhydrous ethanol, sterile deionized water, and EP tubes need to be prepared by the user.

• Introduction to the Reagent Kit

This kit provides a rapid and effective purification method for isolating DNA from various bodily fluids and bacterial culture fluids. It utilizes a silicon-based purification column that selectively adsorbs nucleic acids. With specific buffer solutions, bacterial DNA samples can be extracted within 30 分. The entire purification process does not require toxic reagents such as phenol-chloroform. The extracted DNA can be directly used for downstream experiments like PCR, Southern blotting, その他.

• Experimental Principles and Procedures

• Extraction Process

Before Starting the Experiment:

1. Reagent Buffer B and C may precipitate under low-temperature conditions. We recommend heating at 65℃for 5 分. After the precipitate dissolves, it can be used normally.
2. ご使用の前に, add the specified amount of anhydrous ethanol to Wash Buffer 1as indicated on the bottle label. Mark a check on the label to indicate the addition of anhydrous ethanol.
3. Elution Buffer is a0.1x TE solutioncontaining minimal amounts of EDTA. If EDTA might affect subsequent experiments, it is advisable to substitute Elution Buffer with sterile deionized water.
4. Sample Handling:
5. Take around 1 ml of bacterial culture, で遠心分離する 12,000 の回転数 1 分, aspirate the supernatant as much as possible, 追加 200μl of Reagent Buffer Bto the bacterial cell solution. Generally, residual RNA has minimal impact on downstream experiments. If RNA interference needs to be eliminated, 追加 10μl of RNaseA (10mg/ml) to the mixture, incubate at 37°C for 2 minutes with vortexing during the period.
6. If the sample being processed contains Gram-positive bacteria, 追加 10μl of Lysozyme (10 mg/ml)そして 200μl of Reagent Buffer B. Generally, residual RNA has minimal impact on downstream experiments. If RNA interference needs to be eliminated, 追加 10μl of RNaseA (10mg/ml) to the mixture, incubate at 37°C for 15-30 minutes with vortexing during the period.
7. 追加 10μl of Proteinase K (10 mg/ml), thoroughly invert and mix, digest at 65°C for 2 分. During this period, invert and mix the sample solution 6-7 times until the sample solution becomes clear after digestion.
8. 追加 200μl of Reagent Buffer Cto the lysate and mix. If a white precipitate appears, it can be left to settle; it won’t affect subsequent experiments once the precipitate disappears.
9. 追加 200μl of ethanol, 十分に混ぜる. Some precipitation might occur but won’t affect subsequent experiments.
10. Transfer the obtained liquid into a DNA extraction purification column, leave at room temperature for 2 分, で遠心分離する 12,000 の回転数 30 秒. Discard the collected waste and reinsert the collection tube into the purification column for the next step.
11. 追加 600μl of Wash Buffer 1, で遠心分離する 12,000 の回転数 30 秒, discard the waste, and reinsert the DNA extraction purification column into the holder.

(注記: Ensure ethanol has been added to Wash Buffer 1.)

7. 追加 500μl of Wash Buffer 1 to the DNA extraction purification column, で遠心分離する 12,000 の回転数 2 分, extending the centrifugation time as needed for a drier membrane.
8. Place the DNA extraction purification column (holder) into a new centrifuge tube, open, and heat at 65°C for 2 分. Extend this step as necessary to evaporate ethanol, preventing ethanol residues from affecting downstream experiments.
9. 追加 50-100μl of Elution Bufferonto the column membrane, で遠心分離する 12,000 の回転数 2 分.

(注記: 1. Eluting DNA with 50 μl of Elution Buffer can increase DNA concentration but reduce the total DNA yield. 2. The eluted DNA from the eluate can be reapplied to the DNA extraction purification column, centrifuge again at 12,000 の回転数 2 minutes to enhance DNA yield.)