残留DNA提取试剂盒（碘化钠法）

本产品遵照中国药典记载的碘化钠法，属于宿主细胞残余DNA抽提试剂盒。请用于疫苗和治疗用生物制品中含有的宿主细胞残余DNA的提取。

本试剂盒可以提取样品中含有的极微量的DNA，回收率较高。DNA的提取操作时间为60-90分钟。提取的DNA可以通过Q-PCR法进行定量。

采用该试剂盒提取DNA有两种方案。方案#1和方案#2分别从提纯样品（如纯化蛋白质）和原料样品（如细胞裂解物，一种含有高浓度蛋白质或脂质的溶液）中提取DNA。两种方案的不同点见下表。

【特点】

・遵照中国药典记载的碘化钠法

・高回收率

・单管提取DNA（无需更换管）

【通过碘化钠法提取DNA的原理】

通过碘化钠和N-月桂酰肌氨酸钠，将样品中的蛋白质及脂质等溶解。再添加2-丙醇，使DNA与糖原共同沉淀。此时离液离子的碘化钠及N-月桂酰肌氨酸钠阻碍样品中的蛋白质及脂质等成分沉淀，DNA及糖原发生特异性沉淀。

【试剂盒构成】

【存储】

2-10℃避光保存。

【50次试验所需的附加材料】

<试剂>

1) 2-丙醇 20 mL

2) 无菌蒸馏水 6 mL

<仪器>

1) 微型离心机

2) 涡旋混合器

3) 50x2mL或1.5mL离心管

4) 微量移液器

5) 微量移液器吸头

6) 加热器

【使用前】

• “洗净液A, CP”根据批次不同，溶液颜色有可能存在差异（无色～浅黄色），产品性能没有问题，属正常现象。
• 在碘化钠溶液, CP或N-月桂酰肌氨酸钠溶液, CP产生沉淀物时，请在50℃条件下加热至沉淀物溶解。
• 请使用未受DNA或DNase污染的试剂及器具。
• 如果提取的DNA中残留有碘化钠，将导致无法通过吸光度准确测量DNA浓度。请通过Q-PCR法将DNA定量。

【使用方法#1】

<试剂的调制>

1. 溶液I

在26mL的“碘化钠溶液, CP”容器中添加6mL“无菌蒸馏水”、1mL“N-月桂酰肌氨酸钠溶液, CP”、65µL“糖原溶液, CP”共计三种。利用涡旋混合器充分混合均匀。

备注：在冷藏保存条件下，溶液I稳定状态可保持约5个月。

2. 溶液II

在40 mL“洗净液 B, CP”容器中添加2µL“糖原溶液, CP”。立即使用涡旋混合器充分混合均匀。

备注：

・此时，即使产生白色沉淀物也不会影响DNA的提取效率。请直接使用。

・在冷藏保存条件下，溶液 II稳定状态可保持约1周。在少量使用时，只需调制所需量即可。例如：20mL“洗净液B, CP”中添加1µL“糖原溶液, CP”。

<DNA的提取>

1. 吸取100µL样品加入1.5 mL或2mL离心管。
2. 再添加300µL配制好的溶液I至该离心管，并使用涡旋混合器混合均匀；
3. 将离心管在60℃条件下加热15分钟
4. 取出离心管，加入 400µL 2-丙醇至离心管，并使用涡旋混合器混合均匀。
5. 离心管在室温下静置15分钟。
6. 将离心管进行离心( 10,000 g, 15分钟，室温)。出现白色颗粒。
7. 用移液管移除或小心倾倒管中上清液，直至管中液体容积小于约100µL。

备注:

・请注意不要去除颗粒

・若有颗粒漂浮，再次将离心管进行离心（10,000 g，15分钟，室温）。

・若残留溶液残留在管壁，将离心管倒置于滤纸以移除残留液体。

8. 在离心管中添加1mL溶液II，并使用涡旋混合器混合均匀。请剧烈搅拌使白色颗粒从离心管上剥离。

9．将离心管进行离心( 10,000 g, 5分钟，室温)。

10. 用移液管或小心倾倒的方式尽可能多地移除管中的上清液。

备注:

・请注意不要去除颗粒

・若有颗粒漂浮，再次将离心管进行离心（10,000 g，5分钟，室温）。

・若残留溶液残留在管壁，将离心管倒置于滤纸以移除残留液体。

11. 将颗粒置于加热器、真空干燥器中加热（60℃）使其干燥，或使其自然干燥。

备注：

・若过分干燥的话，将导致DNA重新溶解困难。当颗粒呈潮湿的状态时，停止干燥。

・颗粒中含有DNA和糖原。

・由于溶液II包含可以抑制qPCR的乙醇，管中残留液体过多会降低qPCR效率。

12. 将颗粒在无菌蒸馏水或缓冲液中重新溶解，至Q-PCR。

【使用方法#2】

<试剂的调制>

1. 溶液I-B

在26mL的“碘化钠溶液，CP”容器中添加65µL的“糖原溶液，CP”，利用涡旋混合器充分混合均匀。

备注：在2-10℃避光保存条件下，溶液I-B稳定状态可保持约5个月。

2. 溶液II

在40 mL“洗净液 B，CP”容器中添加2µL“糖原溶液，CP”。立即使用涡旋混合器充分混合均匀。

备注：

・此时，即使溶液II中产生白色沉淀物也不会影响DNA的提取质量。

・在2-10℃避光保存条件下，溶液 II稳定状态可至少保持约1周。制备少量时，只需调制所需量的“糖原溶液，CP”和“洗净液 B，CP”即可。例如，在20 mL“洗净液 B，CP”无菌容器中添加1µL“糖原溶液，CP”。

<样品制备 >

■样品溶液中蛋白质浓度小于2mg/mL时

1. 将下列试剂加入样品溶液至最终浓度。

试剂 最终浓度

十二烷基硫酸钠 0.10%

二硫苏糖醇 50mmol/L

2. 在加热器中加热（55℃）30分钟。

■样品溶液中蛋白质浓度大于2mg/mL时

1. 将下列试剂加入样品溶液至最终浓度。

试剂 最终浓度

十二烷基硫酸钠 0.10%

二硫苏糖醇 50mmol/L

氯化钠 150mmol/L

乙二胺四乙酸 （EDTA） 1mmol/L

2. 将蛋白酶K按照样品溶液中每1mg蛋白质20µg的比例加入样品溶液。
3. 采用三羟甲基氨基甲烷将pH调至约7.5。
4. 在加热器中加热（55℃）至少1小时。

<DNA的提取>

1. 吸取400-500µL 样品溶液加入2mL空白离心管。
2. 添加20µL“N-月桂酰肌氨酸钠溶液，CP”至该离心管，并使用涡旋混合器混合均匀。
3. 添加500µL溶液I-B至该离心管，并使用涡旋混合器混合均匀。
4. 在加热器中将离心管在40℃条件下加热15分钟。
5. 从加热器中取出离心管。加入900µL 2-异丙醇至离心管，并使用涡旋混合器混合均匀。
6. 离心管在室温下静置15分钟。
7. 将离心管进行离心( 10,000 g, 15分钟，室温)。出现白色颗粒。
8. 用移液管移除或小心倾倒管中上清液，直至管中液体容积小于约100µL。

备注：

・请注意不要去除管中颗粒。

・若有颗粒漂浮，再次将离心管进行离心（10,000 g，15分钟，室温）。

・若残留溶液残留在管壁，将离心管倒置于滤纸以移除残留液体。

9. 添加800µL“洗净液A，CP”至该离心管，并使用涡旋混合器充分混合均匀。

确保颗粒从管壁脱落。

10. 将离心管进行离心( 10,000 g, 5分钟，室温)
11. 用移液管移除或小心倾倒管中上清液，直至管中液体容积小于约100µL。

备注：

・请注意不要去除管中颗粒。

・若有颗粒漂浮，再次将离心管进行离心（10,000 g，5分钟，室温）。

・若残留溶液残留在管壁，将离心管倒置于滤纸以移除残留液体。

12. 添加5mL溶液II至该离心管，并使用涡旋混合器充分混合均匀。
13. 将离心管进行离心( 10,000 g, 5分钟，室温)。
14. 用移液管或小心倾倒的方式尽可能多地移除管中的上清液。

备注：

・请注意不要去除管中颗粒。

・若有颗粒漂浮，再次将离心管进行离心（10,000 g，5分钟，室温）。

・若残留溶液残留在管壁，将离心管倒置于滤纸以移除残留液体。

15. 将颗粒置于加热器、真空干燥器中加热（60℃）使其干燥，或使其自然干燥。

备注：

・若过分干燥的话，将导致DNA重新溶解困难。当颗粒呈潮湿的状态时，停止干燥。

・颗粒中含有DNA和糖原。

・由于溶液II包含可以抑制qPCR的乙醇，管中残留液体过多会降低qPCR效率。

16. 将颗粒在无菌蒸馏水或缓冲液中重新溶解，至Q-PCR。

【FAQ】

1. 利用吸光度测量提取的DNA浓度时，在230nm波长处出现波峰。
2. 虽然残留有碘化钠，但对于Q-PCR无影响。

1. DNA的回收率低
2. 在DNA提取操作步骤7.和10.中极有可能一部分颗粒被除去。请使用移液管谨慎去除上清液。

<Reference>

Ishizawa, M., Kobayashi, Y., Miyamura, T. and Matuura, S. (1991) Simple Procedure of DNA isolation from human serum, Nucleic Acids Res., 19, 5792.

Code No. 292-81101 (For 50 tests)

DNA Extractor™ Kit for Residual DNA, CP Method(Sodium Iodide Method)

This product is in compliance with sodium Iodide method and designed for use in extracting residual host cell DNA in biological samples such as vaccines and biopharmaceuticals.

It offers high recovery of DNA even from samples containing only a very small amount of DNA. The entire extraction step is completed with a single tube for 60-90 minutes. The extracted DNA can be quantified by qPCR.

There are two protocols for DNA extraction using this kit. Protocol #1 and Protocol #2 are used for DNA extracting from clean samples such as purified protein and crude samples such as cell lysate, solution containing high-concentration protein or lipid, respectively. Difference between the two protocols are indicated in the table below.

【Features】

・In compliance with sodium Iodide method

・High quality and recovery

・Completed in a single tube for 60-90 minutes.

【Principle of sodium Iodide method for DNA extraction】

An chaotropic reagent, Sodium Iodide, and an anionic detergent participate in solubilization of proteins and lipids contained in biological samples. After addition of 2-propanol to the mixture, nucleic acids are co-precipitated with glycogen as a carrier, while other components remain soluble in the solution phase.

【Kit contents】

【Storage】

Store at 2-10℃ in the dark.

【Additional materials required for 50 tests】

<Reagents>

1) 2-Propanol 20 mL

2) Sterile distilled water 6 mL

<Instruments>

1) Microcentrifuge

2) Vortex mixer

3) 50x2mL or 1.5mL of centrifuge tube

4) Micropipette

5) Micropipette tips

6) Heating block

【Precautions for Use】

• The color of “Washing Solution A, CP”may be slightly different between product lots. g. faint yellow or colorless. It does not affect quality of DNA extraction.
• If precipitate is observed in “Sodium Iodide Solution, CP” or “Sodium N-Lauryl Sarcosinate Solution, CP”, warm it or them at 50℃until the precipitate is no longer visible.
• Use DNA-free, DNase-free and sterilizedreagents and Instruments.
• DNA concentration cannotbe accurately measured by absorbance if sodium Iodide remains in the extracted DNA. Measure the amount of DNA by qPCR

【Protocol #1】

< Preparation of Reagents>

Solution I-A

Add 6mL of sterile distilled water, 1mL of “Sodium N-Lauryl Sarcosinate Solution, CP” and 65µL of “Glycogen Solution, CP” into the bottle of 26mL of “Sodium Iodide Solution, CP”. Mix it with a vortex mixer.

Note : Solution I-A can be stored at 2-10℃ in the dark for approximately 5 months.

Solution II

Add 2µL of “Glycogen Solution, CP” into the bottle of 40 mL of “Washing Solution B, CP”. Mix it with a vortex mixer immediately.

Notes :

• It does not affect quality of DNA extraction if white precipitates appear in Solution II.
• Solution II can be stored at 2-10℃in the dark for approximately a week at least. If preparing a small volume of Solution II, use required amount of “Glycogen Solution, CP” and “Washing Solution B, CP”. For example, add 1µL of “Glycogen Solution, CP” into 20mL of “Washing Solution B, CP” in a sterilized tube.

< DNA Extraction Procedure>

1. Dispense100µof sample solution to a 2mL or 1.5mL blank centrifuge tube.
2. Add 300µLof Solution I-A into the tube and mix it with a vortex mixer.
3. 3.Warm the tube at 60℃ for 15 minutes in a heating block.
4. 4.Remove the tube from the heating block. Add 400µLof 2-Propanol into the tube and mix it with a vortex mixer.
5. Leave the tube at room temperaturefor 15 minutes.
6. Centrifugethe tube with 10,000 g at room temperature for 15 minutes.

A faint white pellet appears in the tube.

7. Remove supernatant from the tubeby using a pipette or decanting carefully until the liquid volume in the tube is less than approximately 100µL.

Notes :

・Be careful not to remove the pellet from the tube.

・Centrifuge the tube with 10,000 g at room temperature for 15 minutes again if the pellet floats.

・In case that residual liquid remains on the tube wall, place the tube upside down on a paper filter to remove it.

8. Add 1mL of Solution IIinto the tube and mix it with a vortex mixer vigorously.

Ensure that the pellet is detached from the tube wall.

9．Centrifuge the tube with 10,000 g at room temperature for 5 minutes.

10. Removeas much supernatant as possible from the tube by using a pipette or decanting carefully.

Notes :

・Be careful not to remove the pellet from the tube.

・Centrifuge the tube with 10,000 g at room temperature for 5 minutes again if the pellet floats.

・In case that residual liquid remains on the tube wall, place the tube upside down on a paper filter to remove it.

11. Dry the pellet by warming it at 60℃ in a heating block, vacuum dryeror natural drying.

Notes :

・Too dry pellet is difficult to be dissolved. Stop drying the pellet when it still is wet.

・Much residual liquid in the tube may cause low qPCR efficiency because Solution II contains ethanol, which can inhibit qPCR.

・The pellet contains DNA and glycogen.

12. Dissolve the pellet with sterile distilled wateror buffer, and proceed with qPCR.

【Protocol #2】

< Preparation of Reagents >

1. Solution I-B

65µL of “Glycogen Solution, CP” into the bottle of 26mL of “Sodium Iodide Solution, CP”. Mix it with a vortex mixer.

Note : Solution I-B can be stored at 2-10℃ in the dark for approximately 5 months.

3. Solution II

Add 2µL of “Glycogen Solution, CP” into the bottle of 40 mL of “Washing Solution B, CP”. Mix it with a vortex mixer immediately.

Notes :

・It does not affect quality of DNA extraction if white precipitates appear in Solution II.

・Solution II can be stored at 2-10℃ in the dark for approximately a week at least. If preparing a small volume of Solution II, use required amount of “Glycogen Solution, CP” and “Washing Solution B, CP”. For example, add 1µL of “Glycogen Solution, CP” into 20mL of “Washing Solution B, CP” in a sterilized tube.

< Preparation of Samples >

■In case that protein concentration in the sample solution is less than 2mg/mL.

2. Addthe reagents below into the sample solution to the final concentration.

Reagents Final concentration

Sodium Dodecyl Sulfate 0.10%

Dithiothreitol 50mmol/L      

2. Warm it at 55℃for 30 minutesin a heating block.

■In case that protein concentration in the sample solution is more than 2mg/mL.

1. Addthe reagents below into the sample solution to the final concentration.

Reagents Final concentration

Sodium Dodecyl Sulfate 0.10%

Dithiothreitol 50mmol/L

Sodium Chloride 150mmol/L

EDTA 1mmol/L      

2. Add 20µg of Proteinase K per 1mg of protein in the sample solution in to the sample solution.
3. Adjust pH to be approximately 7.5 with Tris.
4. Warm it at 55℃for an hour at least in a heating block.

< DNA Extraction Procedure>

17. Dispense 400-500µL of sample solution to a 2mL blank centrifuge tube.
18. Add 20µL of “Sodium N-Lauryl Sarcosinate Solution, CP” into the tube and mix it with a vortex mixer.
19. Add 500µL of Solution I-B into the tube and mix it with a vortex mixer.
20. Warm the tube at 40℃for 15 minutes in a heating block.
21. Remove the tube from the heating block. Add 900µL of 2-Propanol into the tube and mix it with a vortex mixer.
22. Leave the tube at room temperature for 15 minutes.
23. Centrifuge the tube with 10,000 g at room temperature for 15 minutes.

A faint white pellet appears in the tube.

24. Remove supernatant from the tubeby using a pipette or decanting carefully until the liquid volume in the tube is less than approximately 100µL.

Notes :

・Be careful not to remove the pellet from the tube.

・Centrifuge the tube with 10,000 g at room temperature for 15 minutes again if the pellet floats.

・In case that residual liquid remains on the tube wall, place the tube upside down on a paper filter to remove it.

25. Add 800µL of “Washing Solution A, CP” into the tube and mix it with a vortex mixer vigorously.

Ensure that the pellet is detached from the tube wall.

26. Centrifuge the tube with 10,000 g at room temperature for 5 minutes.
27. Remove supernatant from the tubeby using a pipette or decanting carefully until the liquid volume in the tube is less than approximately 100µL.

Notes :

・Be careful not to remove the pellet from the tube.

・Centrifuge the tube with 10,000 g at room temperature for 5 minutes again if the pellet floats.

・In case that residual liquid remains on the tube wall, place the tube upside down on a paper filter to remove it.

28. Add 1.5mL of Solution II into the tube and mix it with a vortex mixer vigorously.
29. Centrifuge the tube with 10,000 g at room temperature for 5 minutes.
30. Removeas much supernatant as possible from the tube by using a pipette or decanting carefully.

Notes :

・Be careful not to remove the pellet from the tube.

・Centrifuge the tube with 10,000 g at room temperature for 5 minutes again if the pellet floats.

・In case that residual liquid remains on the tube wall, place the tube upside down on a paper filter to remove it.

31. Dry the pellet by warming it at 60℃ in a heating block, vacuum dryeror natural drying.

Notes :

・Too dry pellet is difficult to be dissolved. Stop drying the pellet when it still is wet.

・Much residual liquid in the tube may cause low qPCR efficiency because Solution II contains ethanol, which can inhibit qPCR.

・The pellet contains DNA and glycogen.

32. Dissolve the pellet with sterile distilled water or buffer, and proceed with qPCR.

【FAQ】

1. Peak wavelengthappeared at 230nm when extracted DNA concentration was measured by absorbance.
2. Sodium Iodide remains in the extracted DNA. Measure the amount of DNA by qPCR

1. DNA recovery rate was too low.
2. Apart of DNApellet may have been removed at step 7 or 10. Remove the supernatant with a pipette carefully.

<Reference>

Ishizawa, M., Kobayashi, Y., Miyamura, T. and Matuura, S. (1991) Simple Procedure of DNA isolation from human serum, Nucleic Acids Res., 19, 5792.