How do you transfect HeLa cells?
Transfection of HeLa S3 Cells Plate 4 x 104 cells per well in 0.5 ml of complete growth medium. Cell density should be 50~80% confluent on the day of transfection. For each well of cells to be transfected, dilute 0.25 μg of DNA into 100 μl of Opti-MEM® I Reduced Serum Medium without serum.
How to add Lipofectamine to cells?
Add the oligomer-Lipofectamine 2000 complexes to each well containing cells and medium. Mix gently by rocking the plate back and forth. Incubate the cells at 37°C in a CO2 incubator for 24-96 hours until you are ready to assay for gene knockdown. Medium may be changed after 4-6 hours.
What is Lipofectamine LTX?
Lipofectamine® LTX Reagent is an advanced solution for gene expression in both common and challenging cell types. Lipofectamine® LTX Reagent is synthesized from 100% animal origin-free components, making it easy to validate the absence of zoonotic diseases, such as BSE or viruses, in research experiments or cell lines.
Why are HeLa cells used in transfection?
Research with HeLa Cells Telomere shortening, which is implicated in eventual cell death after a number of divisions, is prevented in HeLa by the enzyme telomerase which restores telomeres during mitosis. Since their initiation, HeLa cells have been used in transfection experiments all over the world to study viruses.
Are HeLa cells hard to transfect?
Transfection difficulty is not limited to immature or primary cells. HeLa, one of biology’s workhorse cell lines, is refractory to high transfection efficiency using chemical methods.
What is lipofectamine transfection?
Lipofectamine or Lipofectamine 2000 is a common transfection reagent, produced and sold by Invitrogen, used in molecular and cellular biology. It is used to increase the transfection efficiency of RNA (including mRNA and siRNA) or plasmid DNA into in vitro cell cultures by lipofection.
How long does it take HeLa cells to adhere?
some like HeLa only take a couple of hours, others like primary cells often take 12 hours or more.
How long does it take for u2os cells to attach?
If you trypsinized the adherent cells then replate them probably only need a few minutes for some of them to attach. For both endothelial and sommoth muscle cells I have seen them attached withing 20 minutes whereas from liquid N2 it may take 2-4 hours.
Can you transfect cells twice?
Yes, it can be transfected, in principle. So when you have transfected the stable cells for the second time with the same plasmid, did you observe any increase in the expression of the transgene or any change in the expression copy number?
Can you Trypsinize transfected cells?
Yes, it works fine to trypsinize and re-plate cells 24 hours after siRNA transfection.
What is the role of Lipofectamine?
What is Lipofectamine LTX reagent?
Lipofectamine LTX™ Reagent is a proprietary, animal-origin free formulation for the transfection of DNA into eukaryotic cells with low cytotoxicity. This reference provides a recommended procedure to transfect plasmid DNA into HEK 293, human embryonic kidney cells (ATCC No. CRL-1573) using Lipofectamine LTX™ Reagent.
How do I transfect HEK 293 cells with Lipofectamine LTX™reagent?
Follow these important guidelines when transfecting HEK 293 cells using Lipofectamine LTX™Reagent: • Maintain the same seeding conditions between experiments. Use low-passage cells; make sure cells are healthy and greater than 90% viable before transfection. • Transfection can be performed both in the presence or absence of serum.
What is the protocol for Lipofectamine 3000 reagent?
Lipofectamine™ 3000 Reagent Protocol For Research Use Only. Not for use in diagnostic procedures. Plate cells so they will be 70–90% confluent at the time of transfection.Prepare plasmid DNA-lipid complexes (recommend 2 doses of lipid).
How do you mix Lipofectamine LTX with Opti-Mem?
Aliquot the appropriate amount of the “Master Mix” (equivalent to the optimized amount of Lipofectamine® LTX), and bring up the final volume to 10 μl per well with Opti-MEM® I Reduced Serum Medium. Mix gently. Add the diluted Lipofectamine® LTX to the diluted DNA.