Lipofectamine® 2000 Transfection Reagent

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Add diluted siRNA to diluted Lipofectamine Reagent Incubate for 20 min at RT and add to cells. Replace medium after 5h with 5% heat-inactivated FBS and re-incubate cells at 37°C for 48 h

Protocol tips
Add diluted siRNA to diluted Lipofectamine Reagent Incubate for 20 min at RT and add to cells. Replace medium after 5h with 5% heat-inactivated FBS and re-incubate cells at 37°C for 48 h

Publication protocol

The transfection grade eukaryotic expression vector pUSEamp(+), containing the full-length wild type cDNA of human RhoA gene, was purchased from Millipore. To construct the RhoA expression vector, WT-RhoA cDNA was subcloned into the Xho1 and HindIII sites of an expression vector pcDNA3.1(+) (Invitrogen) with the cytomegalovirus immediate-early promoter, and resulting clones were sequenced for the confirmation of successful subcloning of WT-RhoA cDNA. The IEC-6 cells were transfected with the WT-RhoA expression vector or control vector containing no RhoA cDNA (Null) by using LipofectAMINE 2000 and performed as recommended by the manufacturer (Invitrogen). After the 5-h period of incubation, the transfection medium was replaced by the standard growth medium containing 5% FBS for 2 days before exposure to the selection medium. These transfected cells were selected for RhoA integration by incubation with the selection medium containing 0.6 mg/ml of G418, and clones resistant to the selection medium were isolated, cultured, and screened for RhoA expression by Western blot analysis with the specific anti-RhoA antibody.

Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing siRNA / RNAi /miRNA transfection Rat - IEC-6 Lipofectamine using Lipofectamine® 2000 Transfection Reagent from Thermo Fisher Scientific.

Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for Lipofectamine® 2000 Transfection Reagent below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing siRNA / RNAi /miRNA transfection Rat - IEC-6 Lipofectamine using Lipofectamine® 2000 Transfection Reagent from Thermo Fisher Scientific. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.