Plasmid DNA Transfection Protocol

by: Thermo Fisher Scientific

Download this transcript

Transcript:

[3.83]
in this video we will perform a plasmid DNA transfection experiment using lipo effect Amin LTX and plus we agent 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 lipo effect Amin LTX and Plus reagent optimum 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 1.5 milliliter micro centrifuge tubes in Iraq a pea 200 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 lipo effect Amin LTX and Plus reagent protocol prepare for tubes each with 50 microliters of optimum medium and label them 1 to 4 add 2 microliters of lipo effect Amin 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 optimum medium and add 5 micro grams of plasmid DNA since our DNA concentration is at 1 microgram per microliter we are adding 5 microliters next add 5 microliters of plus reagent and mix well


[125.04]
add 50 microliters of the diluted DNA to each of the lipo effect Amin LTX delusions in tubes 1 2 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 2 4 - Wells 1 2 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 one to three days at 37 degrees Celsius after incubating your cells assess the transfection efficiency in each well by viewing GFP fluorescence examine each well using a Floyd's cell imaging station or microscope to determine which concentration of reagent provided the highest transfection efficiency in this experiment dilution 3 provided the



Description:
More from this creator:
Learn more at http://www.lifetechnologies.com/transfection

Optimized protocol for Lipofectamine LTX & Plus reagent: http://tools.invitrogen.com/content/sfs/manuals/LipofectamineLTX_PLUS_Reag_protocol.pdf

For technical questions, please reach out to Technical Support at: 'techsupport@lifetech.com'

or check http://www.invitrogen.com/site/us/en/home/support/Contact-Us.html

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/transfection

Disclaimer:
TranscriptionTube is a participant in the Amazon Services LLC Associates Program, an affiliate advertising program designed to provide a means for sites to earn advertising fees by advertising and linking to amazon.com
Contact:
You may contact the administrative operations team of TranscriptionTube with any inquiries here: Contact
Policy:
You may read and review our privacy policy and terms of conditions here: Policy