JetPrime

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Always add your plasmid to the buffer before adding JetPrime

Upstream tips
Always add your plasmid to the buffer before adding JetPrime

Publication protocol

DNA Constructs, Lentivirus Preparation, and Generation of Stably STAT3-Transduced LFs
The STAT3C-expressing vector was obtained from the nonprofit plasmid repository Addgene (Cambridge, MA) according to a material transfer agreement with Dr. James Darnell (Rockefeller University, New York, NY).26 The vectors pLenti6/V5-GW/STAT3C was constructed using the ViraPower Lentiviral Expression System (Invitrogen) and the pLenti6/V5 Directional TOPO Cloning Kit (Invitrogen) according to the manufacturer's protocols. pLenti6/V5-GW/LacZ vector was provided by Invitrogen and used as an infection control. The control LFs were infected with lentivirus at an multiplicity of infection of 5, and stably transfected cells were generated using the selective antibiotic blasticidin (Invitrogen) according to the manufacturer's recommendations. Stably expressing pLenti6/V5-GW/STAT3C and pLenti6/V5-GW/LacZ clones of the control LFs were maintained in blasticidin-free media for 72 hours before experimental setup. The ORF clone of human STAT3 DNA expressing in the vector pCMV6Entry was purchased from OriGene (Rockville, MD). HFL-1 cells were transiently transfected with a mix of pCMV6Entry-STAT3 and pcDNA3–green fluorescent protein (GFP) (1:1) plasmids using jetPRIME transfection reagent (Polyplus-Transfection Inc., New York, NY) according to the manufacturer's protocol. Cells transfected with pcDNA3-GFP vector alone were used as an irrelevant vector/transfection control. Efficiency of the transfection protocol was evaluated by counting of GFP-positive cells, and it was >50%. Characteristics of the transfected cells are provided in Supplemental Figure S1, A-C (available at http://ajp.amjpathol.org), demonstrating that approximately 45% of cells are strongly positive for GFP fluorescence in plane cell culture 48 hours after transfection.

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 DNA transfection Mammalian cells - Primary cells Human lung fibroblasts (HLF) using JetPrime from Polyplus transfections.

Manufacturer protocol

Download the product protocol from Polyplus transfections for JetPrime below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing DNA transfection Mammalian cells - Primary cells Human lung fibroblasts (HLF) using JetPrime from Polyplus transfections. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.