本试剂盒适合从细菌样品中快速、简便地提取基因组DNA，提取的基因组DNA片段大、纯度高、稳定性好，可直接用于PCR、酶切、核酸杂交等实验。提取过程无需酚-氯仿抽提，细菌样品经裂解液裂解和蛋白酶K消化后，在无水乙醇的调节下，基因组DNA可与吸附柱WHF特异性结合；经过快速充分的洗涤步骤，可有效去除残留的蛋白质、盐分等杂质；最后，DNA溶解于Buffer EB中，获得高纯度的基因组DNA。
产品号
| 组件
| 物理外观
| 50T
| 储存温度
|
B1522827A
| Buffer UA
| 液体
| 15 mL | RT.
|
B1522827B
| Buffer UL
| 液体
| 15 mL | RT.
|
B1522827C
| Buffer PW1
| 液体
| 17 mL
| RT.
|
B1522827D
| Buffer PW2
| 液体
| 15 mL
| RT.
|
B1522827E
| Buffer EB
| 液体
| 15 mL
| RT.
|
B1522827F
| 溶菌酶消化Buffer
| 液体
| 9 mL
| RT.
|
B1522827G
| 蛋白酶K
| 液体
| 1.2 mL
| RT.
|
B1522827H
| 吸附柱WHF (含2mL收集管)
| —
| 1 EA×50 | RT. |
本试剂盒所有组分可置于室温干燥条件下保存15个月。
注意事项
1. 样品应避免反复冻融，否则会导致提取的DNA片段较小且提取量下降。
2. 如需要去除RNA，需自备试剂RNase A (100mg/mL)，具体使用方法参见对应操作步骤。
3. 实验需自备无水乙醇，用于Buffer PW1和Buffer PW2的预处理；革兰氏阳性菌提取需额外自备溶菌酶溶液 (50mg/mL)（货号L1520264）。
4. 使用前请检查Buffer UA是否出现结晶或者沉淀，如有结晶或者沉淀，请将Buffer UA置于37℃水浴中重新溶解后使用。
操作步骤
使用前请先在Buffer PW1中加入22 mL无水乙醇，Buffer PW2中加入60 mL无水乙醇。
一、革兰氏阴性菌基因组DNA提取
1. 取细菌培养液1-5 mL（含10⁶-10⁹个细胞，最多不超过2×10⁹个细胞）置于自备的离心管中，12,000 rpm (13,680 × g) 离心1 min，尽量吸净上清液，保留菌体沉淀。
2. 向菌体沉淀中加入200 μL Buffer UA，振荡至菌体彻底悬浮。
3. 加入20 μL 蛋白酶K，涡旋混匀，56℃孵育，直至溶液变清亮；孵育过程中每隔一段时间颠倒或振荡离心管，使样本充分分散，促进裂解。
【注意】如果需要去除RNA，可在上述步骤完成后，加入4 μL RNase A (100 mg/mL)溶液，振荡15 sec，室温放置15-20 min。
4. 加入200 μL Buffer UL，涡旋振荡充分混匀；再加入200 μL无水乙醇，涡旋振荡充分混匀；短暂离心，收集管盖内壁上的溶液，避免试剂残留。
【注意】
(1) 如果同时提取多个样品，可以预先将Buffer UL和无水乙醇等比例混合，每管加入400 μL混合液，涡旋振荡充分混匀，提高操作效率；
(2) 加入Buffer UL和无水乙醇后可能会产生白色沉淀，属于正常现象，不影响后续实验。
5. 将步骤4得到的溶液和沉淀一起转入吸附柱WHF（将吸附柱放入配套的2 mL收集管中），12,000 rpm (13,680 × g) 离心30 sec，弃去收集管中的废液，将吸附柱重新放回收集管中。
6. 向吸附柱中加入700 μL Buffer PW1（使用前已加入无水乙醇），12,000 rpm (13,680×g) 离心30 sec，弃去废液，将吸附柱放回收集管中。
7. 向吸附柱中加入700 μL Buffer PW2（使用前已加入无水乙醇），12,000 rpm (13,680×g) 离心30 sec，弃去废液，将吸附柱放回收集管中。
【注意】如需进一步提高DNA纯度，可以重复步骤7。
8. 将吸附柱连同收集管一起，12,000 rpm (13,680 ×g) 离心2 min，彻底去除吸附柱中残留的洗涤液和杂质（避免残留乙醇影响后续DNA洗脱和实验）。
9. 将吸附柱从收集管中取出，转入一个新的1.5 mL离心管中，开盖放置2 min，彻底挥发吸附柱中残留的乙醇。向吸附膜中间部位缓慢悬空滴加50-200 μL Buffer EB，室温放置1 min；12,000 rpm (13,400×g) 离心30 sec，收集离心管中的DNA溶液，可立即用于后续实验，或置于-20℃长期保存。
【注意】
(1) 如果下游实验对pH值或EDTA敏感，可以用灭菌水洗脱。洗脱液的pH值对洗脱效率有很大影响，若用水做洗脱液应保证其pH值在7.0-8.5（可以用NaOH将水的pH值调到此范围），pH值低于7.0时洗脱效率不高。
(2) 如果要提高DNA的终浓度，可以将步骤9所得的DNA洗脱液重新加至吸附膜上，室温放置2min，12,000rpm(13,680×g) 离心30sec。
(3) 如需长期保存，建议使用Buffer EB洗脱并于-20℃长期保存，可有效防止DNA降解。
二、革兰氏阳性菌基因组DNA提取
1. 取细菌培养液1-5 mL（含10⁶-10⁹个细胞，最多不超过2×10⁹个细胞）置于自备的离心管中，12,000 rpm (13,680 × g) 离心1 min，尽量吸净上清液，保留菌体沉淀。
2. 向菌体沉淀中加入160 μL 溶菌酶消化Buffer和40 μL 溶菌酶溶液 (50 mg/mL)（自备），振荡至菌体彻底悬浮，无明显沉淀颗粒。
3. 将离心管置于37℃环境中孵育30 min，充分破坏细菌细胞壁。
4. 加入20 μL 蛋白酶K，涡旋混匀10 sec；再加入200 μL Buffer UL，涡旋振荡充分混匀。
【注意】不要直接将蛋白酶K加入Buffer UL中，需按上述顺序依次添加。
5. 将离心管置于56℃环境中孵育30 min，进一步促进菌体裂解和蛋白质消化。
【注意】如果需要去除RNA，可在上述步骤完成后，加入4 μL RNase A (100 mg/mL)溶液，振荡15 sec，室温放置15-20 min。
6. 加入200 μL无水乙醇，涡旋振荡充分混匀；短暂离心，收集管盖内壁上的溶液，避免试剂残留。
【注意】加入无水乙醇后可能会产生白色沉淀，属于正常现象，不影响后续实验。
7. 将步骤6得到的溶液和沉淀一起转入吸附柱WHF（将吸附柱放入配套的2 mL收集管中），12,000 rpm (13,680 × g) 离心30 sec，弃去收集管中的废液，将吸附柱重新放回收集管中。
8. 向吸附柱中加入700 μL Buffer PW1（使用前已加入无水乙醇），12,000 rpm (13,680×g) 离心30 sec，弃去废液，将吸附柱放回收集管中。
9. 向吸附柱中加入700 μL Buffer PW2（使用前已加入无水乙醇），12,000 rpm (13,680×g) 离心30 sec，弃去废液，将吸附柱放回收集管中。
【注意】如需进一步提高DNA纯度，可以重复步骤9。
10. 将吸附柱连同收集管一起，12,000 rpm (13,680 × g) 离心2 min，彻底去除吸附柱中残留的洗涤液和杂质（避免残留乙醇影响后续DNA洗脱和实验）。
11. 将吸附柱从收集管中取出，转入一个新的1.5 mL离心管中，开盖放置2 min，彻底挥发吸附柱中残留的乙醇。向吸附膜中间部位缓慢悬空滴加50-200 μL Buffer EB，室温放置1 min；12,000 rpm (13,400×g) 离心30 sec，收集离心管中的DNA溶液，可立即使用或-20℃长期保存。
【注意】
(1) 如果下游实验对pH值或EDTA敏感，可以用灭菌水洗脱。洗脱液的pH值对洗脱效率有很大影响，若用水做洗脱液应保证其pH值在7.0-8.5（可以用NaOH将水的pH值调到此范围），pH值低于7.0时洗脱效率不高。
(2) 如果要提高DNA的终浓度，可以将步骤11所得的DNA洗脱液重新加至吸附膜上，室温放置2 min，12,000 rpm(13,680×g) 离心30 sec。
(3) 如需长期保存，建议使用Buffer EB洗脱并于-20℃长期保存，可有效防止DNA降解。
DNA浓度及纯度检测
提取的基因组DNA片段大小，与样品保存时间、操作过程中的剪切力等因素有关。纯化得到的DNA片段可用琼脂糖凝胶电泳和紫外分光光度计两种方法，检测其浓度与纯度。
1. 浓度检测
通过紫外分光光度计测量样品在260 nm处的吸光度（A<sub>260</sub>），确定DNA浓度。若需稀释样品，建议使用Buffer EB进行稀释，用水稀释样本可能导致数值不准确。260 nm处准确的吸光度应在0.1-1.0范围内，以保证检测结果的准确性。换算标准：260 nm处1个单位的吸光度，相当于50 µg/mL双链DNA，或40 µg/mL单链DNA。
2. 纯度检测
通过检测A<sub>260</sub>/A<sub>280</sub>的比值判断DNA纯度，合格的基因组DNA，其A<sub>260</sub>/A<sub>280</sub>比值应在1.7-1.9之间。如果洗脱时未使用Buffer EB，而是使用ddH₂O，比值会偏低，因为pH值和离子存在会影响光吸收值，但并不表示DNA纯度低。
This kit is suitable for rapid and simple extraction of genomic DNA from bacterial samples. The extracted genomic DNA has large fragments, high purity, and good stability, and can be directly used in experiments such as PCR, restriction enzyme digestion, and nucleic acid hybridization. The extraction process does not require phenol‑chloroform extraction. Bacterial samples are lysed with a lysis buffer and digested with proteinase K. Then, in the presence of absolute ethanol, the genomic DNA binds specifically to the WHF spin column. After rapid and thorough washing steps, residual proteins, salts, and other impurities are effectively removed. Finally, the DNA is dissolved in Buffer EB, yielding high‑purity genomic DNA.
| Product No. | Component | Appearance | 50T | Storage |
|---|
| B1522827A | Buffer UA | Liquid | 15 mL | RT | | B1522827B | Buffer UL | Liquid | 15 mL | RT | | B1522827C | Buffer PW1 | Liquid | 17 mL | RT | | B1522827D | Buffer PW2 | Liquid | 15 mL | RT | | B1522827E | Buffer EB | Liquid | 15 mL | RT | | B1522827F | Lysozyme Digestion Buffer | Liquid | 9 mL | RT | | B1522827G | Proteinase K | Liquid | 1.2 mL | RT | | B1522827H | WHF Spin Column (with 2 mL collection tube) | — | 1 EA × 50 | RT |
|
All components of this kit can be stored under dry conditions at room temperature for 15 months.
Precautions
Samples should be protected from repeated freeze‑thaw cycles, which may result in smaller DNA fragments and reduced yield.
If RNA removal is required, the user needs to supply RNase A solution (100 mg/mL). Refer to the relevant procedure step for usage.
The user needs to supply absolute ethanol for pretreatment of Buffer PW1 and Buffer PW2. For Gram‑positive bacteria extraction, lysozyme solution (50 mg/mL) (Cat. No. L1520264) is also required.
Before use, check Buffer UA for crystallization or precipitation. If present, redissolve by placing in a 37°C water bath.
Procedure
Before use, add 22 mL of absolute ethanol to Buffer PW1 and 60 mL of absolute ethanol to Buffer PW2.
I. Genomic DNA Extraction from Gram‑Negative Bacteria
1. Transfer 1–5 mL of bacterial culture (containing 10⁶–10⁹ cells, maximum 2×10⁹ cells) to a user‑supplied centrifuge tube. Centrifuge at 12,000 rpm (13,680 × g) for 1 min. Remove the supernatant completely, retaining the bacterial pellet.
2. Add 200 μL of Buffer UA to the bacterial pellet and vortex until the cells are completely resuspended.
3. Add 20 μL of Proteinase K, vortex to mix, and incubate at 56°C until the solution becomes clear. During incubation, invert or vortex the tube periodically to disperse the sample and promote lysis.
【Note】 If RNA removal is required, after the above step add 4 μL of RNase A solution (100 mg/mL), vortex for 15 sec, and let stand at room temperature for 15–20 min.
4. Add 200 μL of Buffer UL and vortex thoroughly to mix. Then add 200 μL of absolute ethanol and vortex thoroughly to mix. Briefly centrifuge to collect the solution from the cap wall, avoiding reagent residue.
【Notes】
(1) If extracting multiple samples simultaneously, Buffer UL and absolute ethanol can be premixed in equal proportions, and 400 μL of the mixture can be added to each tube and vortexed thoroughly to improve efficiency.
(2) White precipitate may appear after adding Buffer UL and absolute ethanol; this is normal and does not affect subsequent steps.
5. Transfer the solution and precipitate from step 4 into a WHF spin column (placed in the provided 2 mL collection tube). Centrifuge at 12,000 rpm (13,680 × g) for 30 sec. Discard the flow‑through and return the spin column to the collection tube.
6. Add 700 μL of Buffer PW1 (with ethanol pre‑added) to the spin column. Centrifuge at 12,000 rpm (13,680 × g) for 30 sec. Discard the flow‑through and return the spin column to the collection tube.
7. Add 700 μL of Buffer PW2 (with ethanol pre‑added) to the spin column. Centrifuge at 12,000 rpm (13,680 × g) for 30 sec. Discard the flow‑through and return the spin column to the collection tube.
【Note】 To further increase DNA purity, repeat step 7.
8. Centrifuge the spin column together with the collection tube at 12,000 rpm (13,680 × g) for 2 min to completely remove residual wash buffer and impurities (this avoids residual ethanol affecting subsequent DNA elution and experiments).
9. DNA elution
Transfer the spin column to a new 1.5 mL centrifuge tube. Open the cap and let stand for 2 min to allow residual ethanol to evaporate completely. Slowly add 50–200 μL of Buffer EB dropwise to the center of the membrane without touching it. Let stand at room temperature for 1 min. Centrifuge at 12,000 rpm (13,680 × g) for 30 sec. Collect the DNA solution in the centrifuge tube. The DNA can be used immediately for downstream experiments or stored at –20°C for long‑term preservation.
【Notes】
(1) If downstream applications are sensitive to pH or EDTA, sterile water can be used for elution. The pH of the elution buffer significantly affects elution efficiency. If water is used, ensure its pH is between 7.0 and 8.5 (adjust with NaOH). pH values below 7.0 will reduce elution efficiency.
(2) To increase the final DNA concentration, reload the collected DNA eluate from step 9 onto the same membrane, let stand at room temperature for 2 min, centrifuge at 12,000 rpm (13,680 × g) for 30 sec.
(3) For long‑term storage, it is recommended to elute with Buffer EB and store at –20°C to effectively prevent DNA degradation.
II. Genomic DNA Extraction from Gram‑Positive Bacteria
1. Transfer 1–5 mL of bacterial culture (containing 10⁶–10⁹ cells, maximum 2×10⁹ cells) to a user‑supplied centrifuge tube. Centrifuge at 12,000 rpm (13,680 × g) for 1 min. Remove the supernatant completely, retaining the bacterial pellet.
2. Add 160 μL of Lysozyme Digestion Buffer and 40 μL of lysozyme solution (50 mg/mL) (user‑supplied) to the bacterial pellet. Vortex until the cells are completely resuspended with no visible pellet.
3. Incubate the tube at 37°C for 30 min to thoroughly disrupt the bacterial cell wall.
4. Add 20 μL of Proteinase K and vortex for 10 sec. Then add 200 μL of Buffer UL and vortex thoroughly to mix.
【Note】 Do not add Proteinase K directly into Buffer UL; add in the order specified.
5. Incubate the tube at 56°C for 30 min to further promote cell lysis and protein digestion.
【Note】 If RNA removal is required, after the above step add 4 μL of RNase A solution (100 mg/mL), vortex for 15 sec, and let stand at room temperature for 15–20 min.
6. Add 200 μL of absolute ethanol and vortex thoroughly to mix. Briefly centrifuge to collect the solution from the cap wall, avoiding reagent residue.
【Note】 White precipitate may appear after adding absolute ethanol; this is normal and does not affect subsequent steps.
7. Transfer the solution and precipitate from step 6 into a WHF spin column (placed in the provided 2 mL collection tube). Centrifuge at 12,000 rpm (13,680 × g) for 30 sec. Discard the flow‑through and return the spin column to the collection tube.
8. Add 700 μL of Buffer PW1 (with ethanol pre‑added) to the spin column. Centrifuge at 12,000 rpm (13,680 × g) for 30 sec. Discard the flow‑through and return the spin column to the collection tube.
9. Add 700 μL of Buffer PW2 (with ethanol pre‑added) to the spin column. Centrifuge at 12,000 rpm (13,680 × g) for 30 sec. Discard the flow‑through and return the spin column to the collection tube.
【Note】 To further increase DNA purity, repeat step 9.
10. Centrifuge the spin column together with the collection tube at 12,000 rpm (13,680 × g) for 2 min to completely remove residual wash buffer and impurities (this avoids residual ethanol affecting subsequent DNA elution and experiments).
11. DNA elution
Transfer the spin column to a new 1.5 mL centrifuge tube. Open the cap and let stand for 2 min to allow residual ethanol to evaporate completely. Slowly add 50–200 μL of Buffer EB dropwise to the center of the membrane without touching it. Let stand at room temperature for 1 min. Centrifuge at 12,000 rpm (13,680 × g) for 30 sec. Collect the DNA solution in the centrifuge tube. The DNA can be used immediately or stored at –20°C for long‑term preservation.
【Notes】
(1) If downstream applications are sensitive to pH or EDTA, sterile water can be used for elution. The pH of the elution buffer significantly affects elution efficiency. If water is used, ensure its pH is between 7.0 and 8.5 (adjust with NaOH). pH values below 7.0 will reduce elution efficiency.
(2) To increase the final DNA concentration, reload the collected DNA eluate from step 11 onto the same membrane, let stand at room temperature for 2 min, centrifuge at 12,000 rpm (13,680 × g) for 30 sec.
(3) For long‑term storage, it is recommended to elute with Buffer EB and store at –20°C to effectively prevent DNA degradation.
DNA Concentration and Purity Assessment
The fragment size of the extracted genomic DNA is influenced by factors such as sample storage time and shear forces during handling. The purified DNA can be assessed for concentration and purity by agarose gel electrophoresis and UV spectrophotometry.
1. Concentration determination
Measure the absorbance of the sample at 260 nm (A<sub>260</sub>) using a UV spectrophotometer to determine DNA concentration. If dilution is required, Buffer EB is recommended. Dilution with water may lead to inaccurate readings. The absorbance at 260 nm should be kept within 0.1–1.0 to ensure accuracy. Conversion: 1 unit of absorbance at 260 nm corresponds to 50 µg/mL of double‑stranded DNA or 40 µg/mL of single‑stranded DNA.
2. Purity assessment
Evaluate DNA purity by the A<sub>260</sub>/A<sub>280</sub> ratio. For high‑quality genomic DNA, the A<sub>260</sub>/A<sub>280</sub> ratio should be between 1.7 and 1.9. If ddH₂O instead of Buffer EB is used for elution, the ratio may be lower due to the influence of pH and ions on light absorption; this does not necessarily indicate low DNA purity.
ZJ26F0636843
危险品化学品经营许可证（带存储）