Plasmid DNA Transfection Protocol
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- Опубликовано: 28 июл 2024
- Learn more at www.lifetechnologies.com/trans...
Optimized protocol for Lipofectamine LTX & Plus reagent:
tools.invitrogen.com/content/s...
For technical questions, please reach out to Technical Support at: 'techsupport@lifetech.com' or check www.invitrogen.com/site/us/en/... for contact details.
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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.
For transfection protocols, FAQ's, troubleshooting and tips & tricks visit www.lifetechnologies.com/trans...
you are an awesome lecturer... I watch, like and recommended for others, too. keep it up dear!!
Valérie sur RUclips avec ses tubes à essai : c'est un essai réussi!
علم الاحياء الدقيقه رائع
Excellent
As far as I am concerned, Magic™ mRNA transfection reagent is particularly suitable for mRNA delivery, and has shown outstanding transfection efficiency for both adherent cells and suspension cells.
好干净漂亮的实验室！
ikr
Thank you for this video! Very helpful. If I want to do cotransfection with GFP and APC2 plasmids, shall I add both with the plus reagent?
Thank you for your question. Please contact our technical support team at thermofisher.com/askaquestion and of our agents can assist you with your questions.
Hello, I have used this video for my class work. I need to know the explanation why dilution 3 provides the highest transfection efficiency. Thanks
The cells with the maximum fluorescence with the least amount of product used is the most efficient. So while dilution 3 and 4 looks quite similar, 3 uses less products, so it's the best one.
Good ol' pie-pet !
In this series of dilutions a side-by-side comparison is made of dilutions having very slightly variable final concentrations of GRP and Plus Reagents. Would it help to standardize these across the concentration range of Lipofectamine LTX?
*****
Small variances happen due to different volume(s) of Lipofectamine LTX, I am not saying it matters but if it does the dilution scheme could be adjusted to give the side-by-side intended.
When I calculated it as provided by the demonstration I get Plasmid DNA (ug/uL) of 00.094-0.0092; Lipofectamine LTX (%) 1.96-4.76; Plus Reagent (%) 0.94-0.92 (all from tube 1 to 4 respectively.
A simple resolution is possible, since I am new to this area my biggest question is: does a 3% change at the extreme make any difference in the final analysis?
Thank you!
Why is dilution 3 provides the highest resolution than 2 & 4?
It's not resolution it's transfection efficiency. This is like a titration to figure out which concentration works best.
how much should be added from gfp plasmid control?
Thank you Thuraya for your question. The amount of control added would depend on your well format. For a 96 well, 24 well, or 6 well, you would add 100 ng, 500 ng, or 2500 ng, respectively as indicated in our manual linked below.
assets.thermofisher.com/TFS-Assets/LSG/manuals/LipofectamineLTX_PLUS_Reag_protocol.pdf
For additional technical support, please contact us at Thermofisher.com/askaquestion. Thank you!”
Hey a very useful video I must say, I am however confused to how you add 5 microliters of the DNA. If the DNA was 1 mg/ul why add 5 ul? so what if I did a maxi prep and got 250ng/ul i.e. 0.25mg/ul then how much do I add? what is the dilution and how to you work it out. Please help I am confused.
+Vinay A.K.A convert ng to mg, then use M1V1=M2V2 formula to calculate how much volume you need
The DNA was 1 mg/mL which is equivalent to 1 ug/uL
since we want 5ug of DNA thats 5uL. Maybe because you wrote mg/uL instead of mg/mL that's maybe why you may have been confused
should media be without FBS? or doesn't matter?
with FBS
Is this stable transfection?
Hi Velvet, Thank you for your question. This is a transient transfection.
For additional technical support questions regarding stable transfection, please contact us at thermofisher.com/ask a question. Thank you!”
What does LTX stands for？
Thank you Zou Yu for your question.
For Lipofectamine LTX, it’s the name of the product line.
It’s developed for transfection of primary, challenging, and sensitive cell lines. For additional technical support, please contact us at thermofisher.com/askaquestion.
Thank you!
Which cell line was that??
Thank you for your question.
We actually have newer reagents that we recommend for plasmid DNA transfection and we have a list available of cell lines and the reagents we recommend for them. I’ve linked to it below.
www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-support/transfection-selection-tool.html
If you have any additional technical questions, please contact our technical support team at thermofisher.com/askaquestion. Thank you!
I think it was eukaryotic cells of some sort
Cell-fec-tine Miss Kana Kana
Cell-fec-tine Miss Annabella
Cell-fec-tine yan Donna Donna
Jodi and Rebecca
3:07 what is she using
Thanks for watching, will get back to you with an answer shortly.
A LifeTechnologies microscope. These are usually built with a setting that can visualize GFP or RFP expression.
@@jordanjennings1375 thanks
If they delivered our own rna/dna I would ageee with this but sadly its gmo rna & dna