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Plasmid DNA Transfection Protocol
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Optimized protocol for Lipofectamine LTX & Plus reagent:
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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.
----------
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.
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