为什么老转不进质粒？？SOS

我的脂染步骤如下，为什么转了n次老转不进去？请老师指教！

1.在无菌新管中用HBS稀释10μg质粒到0.1μg/μl（终体积为100μl）

2.在另一无菌管中取60μl DOTAP，加入HBS到终体积为200μl 。

3.将100μl 质粒移入DOTAP管中，温和的吹吸几次，混匀转染混合物。

4.25℃孵育转染混合物15min。

5.孵育过程中取准备好的细胞A、B倒掉上清加1640培养基洗涤一次，在细胞A、B中加入含10%小牛血清的1640培养基6ml。

6.将孵育好的DOTAP/质粒混合物分别加入两方瓶中，轻轻摇匀或吹匀,CO2孵箱孵育7（7-22小时基本都试过了）。

7.弃旧培养基后再在两瓶中各加入与前相同的培养基，CO2孵箱放置48小时。

结果无论是测瞬时表达还是用G418筛选（半个月、一个月都试过）测稳定表达都只有20%左右，细胞和质粒都是别人转过，没问题的！最近看《分子克隆实验指南》第三版上说可能是因为血清会强力抑制转染，不知道这种说法是否站得住脚？！

I think serum will definitely interfere with the liposomal transduction. GIBCO has an optimized medium for Lipofectamine. Usually you need to reduce the serum concentration for the culture medium immediately after transfection. However, Efectene (Qiagen) is specially designed to avoid serum interference, therefor they said you can use normal medium. I do not know the reagent you are using. However, I think it is better to reduce the serum concentration for a while after transfection.



Another factor is the cell type. Some cells are refractory to liposomal transduction. It is very possible that for you cells, 20% is the best you can get. If this is the case you may want to consider some other ways. Actually 20% is not that bad. If you can select them with G418, you will easily get enough cells for your experiment.

表达都只有20%左右，这似乎已经不错了。一般来说瞬时表达不会太高；稳定表达应该是100%的。

好像invorgeon的脂转要换无血清的培养液吧

有些国产的也有不用的，但是转染效率不高，而且细胞死亡较多。