Plasmid DNA Transfection Protocol скачать в хорошем качестве

Plasmid DNA Transfection Protocol

Learn more at http://www.lifetechnologies.com/trans... Optimized protocol for Lipofectamine LTX & Plus reagent: http://tools.invitrogen.com/content/s... For technical questions, please reach out to Technical Support at: '[email protected]' or check http://www.invitrogen.com/site/us/en/... for contact details. ---------- Audio transcript: How to perform Plasmid DNA transfection with Lipofectamine® LTX and Plus™ Reagent protocol. Superior plasmid delivery and protein expression. In this video, we will perform a plasmid DNA transfection experiment using Lipofectamine® LTX & Plus™ reagent. As always, use good cell culture practices and wear your personal protective equipment. Be sure to clean your cell culture hood and work surface by spraying and wiping them down with 70% ethanol. The day prior to your transfection, seed your cells so that they will be 70% to 90% confluent at the time of your experiment. For this transfection experiment you will need: Lipofectamine® LTX and Plus™ Reagent Opti-MEM® Reduced-Serum Medium Plasmid DNA at 1 microgram per microliter. We will be using a Green Fluorescent Protein plasmid to serve as a positive control for transfection. Five, 1.5 mL microcentrifuge tubes in a rack A P200 and P10 pipette and appropriate tips A marker and a timer And a 24-well plate with 70% to 90% confluent cells. We will be following the 24-well plate format of the Lipofectamine® LTX & Plus™ Reagent protocol. Prepare 4 tubes each with 50 microliter of Opti-MEM® Medium, and label them 1 to 4. Add 2 microliters of Lipofectamine® LTX Reagent to tube 1, 3 microliters to tube 2, 4 microliters to tube 3 and 5 microliters to tube 4. Mix each tube well by vortexing or flicking the tube. Prepare a tube with 250 microliters of Opti-MEM® medium and add 5 micrograms of plasmid DNA. Since, our DNA concentration is at 1 microgram per mircoliter we are adding 5 microliters. Next add 5 microliters of Plus™ Reagent and mix well. Add 50 microliters of the diluted DNA to each of the Lipofectamine® LTX dilutions in tubes 1 to 4. Incubate the complex for 5 minutes at room temperature. After the 5-minute incubation, remove your 24-well plate containing your cells from the incubator and bring it to the workspace in the hood. Add 50 microliters of the DNA-reagent complex from tubes 1 to 4 to wells 1 to 4 of the 24-well plate, respectively. You should have enough volume to run duplicates on the same plate if desired. Place your 24-well plate back into the incubator and grow cells for 1 to 3 days at 37 Celsius. After incubating your cells, assess the transfection efficiency in each well by viewing GFP fluorescence. Examine each well using a FLoid cell imaging station or microscope to determine which concentration of reagent provided the highest transfection efficiency. In this experiment dilution 3 provided the highest transfection efficiency. For transfection protocols, FAQ's, troubleshooting and tips & tricks visit http://www.lifetechnologies.com/trans...