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磷酸钙法细胞转染试剂盒
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为什么选择此级别
BioReagent,分子生物学级,无菌 级 ，适用于对基线干扰要求严格的色谱和分析工作流程。
储存与运输
2-8°C储存,-20°C储存。超低温运输 。请查阅批次 COA 获取详细规格。
质量文档
SDS、COA、产品数据表及规格说明书均可下载。可通过批号查询获取批次 COA。
文献证明
在色谱分析、有机合成和交叉偶联反应领域已被 0 篇同行评审文献引用。
概述
外源基因导入真核细胞的方法有很多种，如磷酸钙转染法、DEAE-葡聚糖转染法、脂质体法、电穿孔法、显微注射法等。磷酸钙法细胞转染试剂盒 (Calcium Phosphate Cell Transfection Kit)是在传统的磷酸钙细胞转染方法的基础上进行了改良，提高了转染效率，并降低了毒性，可用于磷酸钙法转染细胞，不仅可以瞬时表达，也可以筛选稳定株，HEK293是最适合磷酸钙法转染的细胞之一，优化条件后转染效率可以高达85%以上，一般的转染效率可达40-50%左右；其他常见细胞 (例如Hela、CHO细胞等)也适合磷酸钙法转染，但效率比293细胞要略低一些。
磷酸钙法细胞转染试剂盒主要适合于大多数贴壁细胞的转染，也可于一些悬浮细胞的转染，一般要求DNA浓度在10-50 μg为宜，Hela、BALB等细胞沉淀放置16h，CHO、DUKX、BⅡ等细胞可以通过甘油、DMSO进行热休克处理以提高转染效率。该试剂盒仅用于科研领域，不适用于临床诊断或其他用途。
自备材料：
胰蛋白酶消化液、完全培养基、PBS、无菌水
操作步骤 (仅供参考)：
(一) 贴壁细胞转染：
1、在转染前24h用胰蛋白酶消化培养细胞，取适量对数期细胞转移至新的培养器皿中，待细胞密度达70-80%即可进行转染。后续操作步骤均按6孔板计算，如果转染器皿不同，请按比例自行调节用量。
2、在加入DNA之前2-4h，加入2 ml不含抗生素的完全培养液，置于37 ℃ 5% CO₂培养箱培养。
3、取2-6 μg DNA (体积不宜超过20 μl)加入100 μl氯化钙溶液，混匀，即为DNA-CaCl₂溶液。
4、取BBS溶液100 μl，用移液器一边吹打BBS溶液，一边逐滴加入DNA-CaCl₂溶液 (操作缓慢，一般在1-2 min)。
5、室温静置20-30 min，即为DNA-CaCl₂-BBS溶液，此时可能出现极其微小颗粒沉淀。
6、取DNA-CaCl₂-BBS溶液底部物质均匀加入到6孔板细胞中，轻轻晃动混匀。
7、置于37 ℃ 5% CO2培养箱培养4-16 h；如果培养细胞为CHO、DUKX等，可以DMSO或甘油进行休克处理，转染效率会大大增加，即培养4-6h后，用2 ml含10%甘油或20%DMSO的完全培养液替换当前培养液，室温下静置3 min，加5 ml PBS摇动混匀。
8、去除培养液，用PBS清洗细胞2次，加入2 ml完全培养液继续培养，一般24h后可见转染细胞的表达。
(二) 悬浮细胞转染：
1、低速离心收集悬浮细胞，用PBS洗涤1次。
2、取2-6 μg DNA (体积不宜超过20 μl)加入100 μl氯化钙溶液，混匀，即为DNA-CaCl₂溶液。
3、取BBS溶液100 μl，用移液器一边吹打BBS溶液，一边逐滴加入DNA-CaCl₂溶液 (操作缓慢，一般在1-2 min)。
4、室温静置20-30 min，即为DNA-CaCl₂-BBS溶液，此时可能出现极其微小颗粒沉淀。
5、每10⁶个细胞沉淀用100μl DNA-CaCl₂-BBS溶液重新悬浮，室温放置20-30 min。
6、6孔板每孔加入2 ml不含抗生素的完全培养基，取DNA-CaCl₂-BBS溶液底部物质均匀加入到6孔板中，轻轻晃动混匀。
7、置于37 ℃ 5% CO₂培养箱培养4-16h，去除培养液，用PBS清洗细胞2次，加入2 ml完全培养液继续培养，一般24h后可见转染细胞的表达。
注意事项：
1、注意无菌操作，尽量避免污染，同时DNA不应含有蛋白和酚。
2、休克处理某些细胞系会使转染效率大大提高，但应注意甘油暴露过久易导致细胞死亡。
3、转染12-24h后，可以加入终浓度为10 mmol/L的丁酸钠溶液，可以提高病毒滴度。
4、BBS溶液的pH值直接关系到转染效率，尽量避免长时间暴露在空气中，以免被空气中的二氧化碳酸化。
5、试剂开封后请尽快使用，以防影响后续实验效果。
6、为了您的安全和健康，请穿实验服并戴一次性手套操作。
There are many methods for introducing foreign genes into eukaryotic cells, such as calcium phosphate transfection, DEAE-dextran transfection, lipofection, electroporation, microinjection, etc. The Calcium Phosphate Cell Transfection Kit is improved based on the traditional calcium phosphate transfection method, with higher transfection efficiency and lower cytotoxicity. It can be used for calcium phosphate-mediated cell transfection for both transient expression and stable cell line selection. HEK293 is one of the most suitable cell lines for calcium phosphate transfection. Under optimized conditions, transfection efficiency can reach above 85%, and typically ranges from 40% to 50%. Other common cell lines (such as HeLa, CHO, etc.) are also compatible but with slightly lower efficiency than 293 cells.
This kit is suitable for transfection of most adherent cells and some suspension cells. The recommended DNA concentration is generally 10-50 μg. Cell lines such as HeLa and BALB can be incubated for 16 h with precipitate; cell lines such as CHO, DUKX, and BⅡ can be subjected to heat shock with glycerol or DMSO to improve transfection efficiency.This kit is for research use only and not for clinical diagnosis or other applications.
Materials Provided by User
Trypsin digestion solution, complete culture medium, PBS, sterile water
Protocol (For Reference Only)
(I) Transfection of Adherent Cells
1. 24 h before transfection: Digest cultured cells with trypsin, seed an appropriate number of log-phase cells into a new culture vessel. Transfection can be performed when cell confluency reaches 70-80%.All procedures below are calculated for a 6‑well plate. Adjust volumes proportionally for other culture vessels.
2. 2-4 h before adding DNA: Add 2 mL of antibiotic‑free complete medium and incubate at 37 ℃ in a 5% CO₂ incubator.
3. Add 2-6 μg DNA (volume≤ 20 μL) to 100 μL Calcium Chloride Solution, mix well to form DNA‑CaCl₂ solution.
4. Take 100 μL BBS Solution. While gently pipetting the BBS Solution, dropwise add the DNA‑CaCl₂ solution (slow operation, about 1-2 min).
5. Incubate at room temperature for 20-30 min to form DNA‑CaCl₂‑BBS solution. Very fine precipitate may appear.
6. Take the mixture (including fine precipitate at the bottom) and add evenly to the cells in the 6‑well plate. Mix gently by shaking.
7. Incubate at 37 ℃ in a 5% CO₂ incubator for 4-16 h.For cell lines such as CHO and DUKX, shock treatment can greatly improve efficiency: After 4-6 h incubation, replace medium with 2 mL complete medium containing 10% glycerol or 20% DMSO, incubate at room temperature for 3 min, then add 5 mL PBS and mix gently.
8. Remove the medium, wash cells twice with PBS, add 2 mL complete medium and continue culture. Transgene expression is usually visible after 24 h.
(II) Transfection of Suspension Cells
1. Collect suspension cells by low‑speed centrifugation, wash once with PBS.
2. Add 2-6 μg DNA (volume≤ 20 μL) to 100 μL Calcium Chloride Solution, mix well to form DNA‑CaCl₂ solution.
3. Take 100 μL BBS Solution. While gently pipetting the BBS Solution, dropwise add the DNA‑CaCl₂ solution (slow operation, about 1-2 min).
4. Incubate at room temperature for 20-30 min to form DNA‑CaCl₂‑BBS solution. Very fine precipitate may appear.
5. Resuspend every 1×10⁶ cells with 100 μL DNA‑CaCl₂‑BBS solution, incubate at room temperature for 20-30 min.
6. Add 2 mL antibiotic‑free complete medium per well of a 6‑well plate. Add the DNA‑CaCl₂‑BBS mixture evenly and mix gently.
7. Incubate at 37 ℃ in a 5% CO₂ incubator for 4-16 h. Remove medium, wash cells twice with PBS, add 2 mL complete medium and continue culture. Transgene expression is usually visible after 24 h.
Precautions
1. Perform aseptic operations to avoid contamination. DNA should be free of protein and phenol.
2. Shock treatment can greatly improve transfection efficiency in some cell lines, but prolonged glycerol exposure may cause cell death.
3. 12-24 h after transfection, adding sodium butyrate to a final concentration of 10 mmol/L can increase viral titer.
4. The pH of BBS Solution directly affects transfection efficiency. Avoid prolonged exposure to air to prevent acidification by CO₂.
5. Use the reagent promptly after opening to avoid affecting experimental results.
6. For your safety and health, wear a lab coat and disposable gloves during operation.
规格
英文别名
Calcium Phosphate Transfection Kit
规格或纯度
BioReagent,无菌,分子生物学级
英文名称
Calcium Phosphate Cell Transfection Kit
应用
Cell Transfection
储存与运输
储存条件
2-8°C储存,-20°C储存
运输条件
超低温运输
稳定性与储存
各组分在相应的储存条件下保质期1年。
组分与储存
| C1509487 | Component | 200T | Storage |
| C1509487A | 氯化钙溶液 | 20 mL | 2-8℃. |
| C1509487B | BBS溶液 | 20 mL | -20℃. |
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F0434118 |
分析证书 | C1509487 |
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