【求助】细胞转染问题

最新回复

• nn255 (2014-12-05 16:08:43)

  
  转染是针对细胞来说的，细胞数，都是在六孔板细胞数不变，转染试剂的配比不用减半。培养基减半的原因是让转染试剂和DNA的复合物与细胞接触的概率更大，更少地悬浮在培养液里。但是正常培养时6孔板一般还是要2ml的吧，不知你培养的是什么细胞呢？为何只用1ml？

• nn255 (2014-12-05 16:09:00)

  还有不知道你用的这是什么转染试剂呢？我们实验室常用的是非脂质体的POLO3000

• zhihui小新 (2014-12-05 16:09:33)

  QUOTE:

  原帖由 zhihui小新 于 2014-12-5 16:08 发表 bbcodeurl('http://bbs.antpedia.com/images/common/back.gif', '')
  转染试剂说明书中（X-treme Gene siRNA Transfection Reagent）六孔板2mL培养基推荐10μL转染试剂稀释至100μL，siRNA稀释至100μL，各孔加转染复合物200μL，各孔终体积为2.2mL。实际操作时，我六孔板每孔为1mL，那么我转染试剂减 ...

  好的，明白了，用1mL是为了后续加药诱导实验，我要先干扰，再诱导，另外1mL在转染时可以节约siRNA，因为siRNA的终浓度是50nM，1mL的话每孔只需20μM的siRNA2.5μL，如果2mL的话就要5μL了，有点浪费。转染试剂是罗氏的，对细胞毒性较小，货号04476093001

• vvmmoy (2014-12-05 16:10:09)

  
  最近我用了一个很好的转染试剂，主要是操作方便，只需将质粒和转染试剂混匀后加到培养基中（不需要更换），且毒性较小，转染效率高，最近那家公司还有免费的试用装

• qhyu (2014-12-05 16:10:34)

  SignaGen公司的GenJet Ver.II，LipoD293及PolyJet针对哺乳动物细胞的DNA体外转染试剂，均为您提供两种不同的转染步骤——一般步骤及高级步骤，这两种步骤分别针对不同的哺乳动物细胞。高级步骤主要针对难转的哺乳动物细胞，例如：MDCK，MDA-MB231，Caco-2等细胞。使用一般的转染步骤若是转染效率低于5%，那么使用高级转染步骤则可以将转染效率提升至60%。
  如何选择转染步骤则取决于您的细胞特性。对于诸如HEK293，Hela，CHO，3T3，COS，HepG2，LNCaP，PC3，PC12，U2-OS，L929，MCF-7及Huh-7等常用细胞，一般的实验步骤就能得到满意的转染结果。对于诸如MDCK，MDA-MB231，Caco-2，SaoS-2及HUVEC等难转细胞，可以选用高级转染步骤。针对您的细胞，若是您尚不明确选择哪种转染步骤，我们建议您可先尝试一般步骤，如果您使用一般步骤得到的转染效率低于5%，那么您的细胞比较难转，您可尝试高级转染步骤。
  Choose a more efficient protocol for your specific cell.
  GenJet Ver. II, LipoD293 and PolyJet DNA in vitro transfection reagent all offer two protocols for transfecting mammalian cells--------a general protocol and an advanced protocol. The two protocol are written for transfecting different types of mammalian cells. The advanced protocol which involves a shaving cell technology is for very hard to transfect mammalian cells like MDCK, MDA-MB231, Caco-2, etc. The general protocol usually gives less than 5% efficiency while advanced protocol can get up to 60% efficiency on these problematic cells.
  The principle to decide which one protocol is used to your cell is highly dependent upon the nature of your cell. For commonly used cells like HEK293, Hela, CHO, 3T3, COS, HepG2, LNCaP, PC3, PC12, U2-OS, L929, MCF-7 and Huh-7, be sure to use general protocol which usually gives very good transgene expression while the advanced protocol does not help. For very hard to transfect cells like MDCK, MDA-MB231, Caco-2, SaoS-2 and HUVEC, follow the advanced protocol by trypsinizing these adherent cells first followed by incubation of the cell pellet with transfection complex. If you do not know for your specific cells how to choose the more efficient protocol, we suggest you try general protocol first. If you get less than 5% efficiency with the general protocol, then your cell is hard to transfect in nature and you can consider trying advanced one.
  可以试用一下！

• nut6694 (2014-12-05 16:10:59)

  
  你好楼主，我也用的罗氏转染试剂，我想问一下，在转染时是不是可以直接将转染复合物添加到六孔板里，不用在转染前换液吧？