产品描述

Hieff Trans^®脂质体核酸转染试剂是一种多用途的脂质体转染试剂，适用于DNA、RNA和寡核苷酸的转染，对大多数真核细胞具有很高的转染效率。其独特的配方使其可直接加入培养基中，血清的存在不会影响转染效率，这样可以减少去除血清对细胞的损伤。转染后不需要除去核酸–Hieff Trans^®复合物或更换新鲜培养基，也可在4～6小时后除去。

Hieff Trans^®脂质体核酸转染试剂以无菌的液体形式提供。通常情况下对于 24 孔板转染，每次用1.5 μL左右，则1 mL 约可做660 次转染；对于6孔板，每次用6 μL左右，则1 mL约可做160 次转染。

运输与保存方法

冰袋（wet ice）运输。产品2-8ºC保存，一年有效。不可冷冻！

注意事项

1. Hieff Trans^®脂质体核酸转染试剂要求细胞铺板密度较高，以60%-80%为佳，这有助于减少阳离子脂质体细胞毒性造成的影响，具体铺板密度需要预实验摸索；如果你研究的基因要求比较长的表达时间，比如细胞周期相关基因，或者细胞表面蛋白，最好选择细胞铺板密度要求较低的转染试剂，不适合用脂质体核酸转染试剂。

2. Hieff Trans^®脂质体核酸转染试剂可用于有血清培养基的转染，并且转染前后不需要换培养基。但是，制备转染复合物时要求用无血清培养基稀释DNA和转染试剂，因为血清会影响复合物的形成。另外，要检测所用的无血清培养基与脂质体核酸转染试剂的相容性，已知CD293, SFMII, VP-SFM就不相容。

3. 转染的时候培养基中不能添加抗生素。

4. 使用高纯度的DNA或RNA有助于获得较高的转染效率，质粒中的内毒素是转染的大敌。

5. 阳离子脂质体应该在2-8ºC保存，要注意避免多次反复长时间开盖，因为可能会导致脂质体氧化而影响转染效率。

6. 初次使用应优化DNA浓度和阳离子脂质体试剂量以得到最大的转染效率。DNA和转染试剂的比例，通常推荐是1:2-1:3，比如24孔板内接种0.5-2×10⁵个细胞，使用0.5 µg DNA和1-1.5 µL 转染试剂。通过调整DNA/Hieff Trans^®脂质体核酸转染试剂比例优化转染效率，DNA（μg）: 试剂（μL）比值在1:0.5-1:5。

7. 本产品仅作科研用途！

操作流程（以24孔板为例，其他培养板加样体积请参考表一） 

【注】：转染试剂使用量受细胞类型及其他实验条件影响，建议初次使用时设置梯度进行优化最佳使用量。

贴壁细胞：转染前一天（20-24小时），胰酶消化细胞并计数，细胞铺板（不含抗生素），使其在转染时密度为70-95%（0.5-2 × 10⁵ cells/well for a 24-well plate）。

悬浮细胞：转染当天，配制DNA复合物之前，24孔板中细胞铺板，每500 µL生长培养基（不含抗生素）中加入4-8 × 10⁵ cells。

1. 按照以下体系配制DNA-Hieff Trans^®脂质体核酸转染试剂复合物：

1）对于每孔细胞，使用50 μL无血清培养基（如OPTI-MEM Ⅰ培养基）稀释0.5 μg DNA。混匀。

2）对于每孔细胞，使用50 μL无血清培养基（如OPTI-MEM Ⅰ培养基）稀释0.6-2.5 μL Hieff Trans^®脂质体核酸转染试剂。

Hieff Trans^®脂质体核酸转染试剂稀释后室温孵育5 min（在30 min内同稀释的DNA 混合，保温时间过长会降低活性）

【注意】：即使脂质体核酸转染试剂使用OPTI-MEM Ⅰ稀释，细胞也可以使用DMEM培养。如果DMEM作为脂质体核酸转染试剂的稀释液，必须在5 min内同稀释的DNA混合。

2. 混合稀释的DNA和稀释的脂质体核酸转染试剂（总体积100 µL），轻轻混匀，并在室温（15-25℃）孵育20 min，使得DNA-脂质体复合物形成。此时溶液可能会混浊，但不会影响转染。

【注意】：DNA-脂质体复合物室温至少稳定保存5 h。

3. 直接将100 µL DNA-Hieff Trans^®复合物加入到细胞培养板每个孔中，摇动培养板，轻轻混匀。

【注意】：如果在无血清条件下转染，使用含血清的正常生长培养基进行细胞铺板。在加入复合物前移去生长培养基，替换为500 µL无血清培养基。

4. 37℃，5% CO₂培养箱培养24-48 h，直至进行转基因表达分析，无需去掉复合物或更换培养基。然而，可能有必要在4-6 h后更换生长培养基，不会降低转染活性。

稳转细胞株：转染24 h后，按照1:10或更高比例在细胞中加入新鲜生长培养基，转染48 h后加入筛选培养基。

悬浮细胞株：在细胞中加入DNA-Hieff Trans^®复合物后，如果需要可以4 h后加入PMA和/或PHA。对于Jurkat细胞，PHA和PMA的终浓度分别为1 µg/mL和50 ng/mL，可以提高CMV启动子活性和基因表达。对于K562细胞，只加入PMA足以提高启动子活性。

转染体系的调整

对于不同的细胞培养板，Hieff Trans^®脂质体核酸转染试剂、DNA、细胞和培养基的使用量会有所不同，具体请参考下表（表一）。对于96 孔板培养，不需要提前一天进行细胞铺板，可以直接在平板中制备复合物，然后将细胞悬浮液加入到复合物就可以了，这样进一步减少了转染时间。这种改进步骤已经过293-H，293-F，COS-7L和CHO细胞的试验，同传统方法相比活性稍低。快捷的步骤和蛋白表达细胞系的高效转染使得脂质体核酸转染试剂非常适用于96 孔板的高通量转染，比如cDNA文库的筛选和蛋白瞬时表达。

表一 在不同的培养容器中转染，脂质体核酸转染试剂，核酸，细胞和培养基的用量

1 不同厂商提供的细胞培养板表面积可能有所不同；

2 稀释DNA或RNAi所用的培养基体积。

【注】：该表使用量仅供参考，具体使用量还需根据细胞类型及其他实验条件进行优化。使用时DNA（μg）: Hieff Trans^®（μL）比值保持在1:0.5-1:5

相关产品

HB220930

Q：转染试剂毒性相比之前的批次大？

A：40802产品进行的工艺优化，纯度增高，相应的转染效率也随之变高，建议质粒与转染试剂的比例在1:2进行调整，一旦出现细胞死亡的现象，降低转染试剂比列。或者转染6h后进行换液。

Q: 它的大致成分和脂质体粒径，我们可以提供吗？

A: 提供不了粒径取决于客户的核酸和实验条件的 不是一个绝对值的。

Q：是稳转特制的转染试剂吗？若不是特制的转染试剂，那是特制的质粒才能进行稳转吗？

A：不是稳转特制的转染试剂，普通的转染试剂。质粒要求：稳转的质粒是普通的质粒，只是需要带有抗性，便于后期的筛选。建议：其中质粒转染受制于质粒大小、转染介质的限制，对很多细胞转染效率低，而且质粒整合入细胞基因组的效率极低，所以构建稳转株的成功率不高，请知悉，若做稳转细胞株，建议进行慢病毒包装（货号：41102）。

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Q：脂质体转染的效率多少，毒性如何？

A：有些细胞如 293T、293FT、Hela 等转染效率基本在85%以上；所有阳离子脂质体转染试剂对细胞都会存在一定的毒性，但是我们公司的转染试剂经过配方优化后其毒性大大降低，且转染效率也有进一步提升。

Q：转染试剂转染后需要换液吗？

A：对于换液可以区分两种情况；1、转染之前如果没有换液应在转染 6 小时左右后换液，以保证细胞生长所需营养，2、如果转染之前如果有换液，可以按照平时等到培养基出现营养不足时换液。

Q：转染试剂转单个质粒和多质粒共转的效率如何？

A:单转效率对于验证过的细胞效率都是很好，可以参考FAQ-验证过的细胞系，对于共转由于要涉及到质粒的混合比例和质粒与转染试剂的添加比例问题，因此具体的效率需要做相应的验证。

Q：转染试剂可以冻存吗？

A:不可以冻存，因为转染试剂是一种脂质体阳离子转染试剂，由于脂质体是不能在低温下冻存，因此转染试剂最好是 4 度储存，保持最好的转染效能。

Q：转染实验过程中是否需要更换成无血清培养基？

A：不需要，我们的转染试剂可以在含血清的介质中进行转染的过程。

Q：转染后需要进行终止反应吗？

A：不需要。脂质体复合物可以稳定存在 6 个小时。如果在进行转染前没有进行细胞换液，为了保证细胞正常生长所需的营养，需要在 4～6 小时后换用新的培养基。但如果转染之前已进行过换液则在脂质体转染后不需要进行再次换液。