Can anyone help me with the Hayflick limit of HEPG-2 cell lines ?

I am testing some PCR primers I designed and I'm having some multiple-banding and sequencing issues with them. I wanted to BLAST them to see what else they may be priming besides the intended target. Everything I have read on Primer-BLAST says to check the specificity primers simply input both primer sequences, choose a database and click "get primers". When I do this I get the error:

Specified left primer not in Included Region; Specified right primer not in Included Region.

I have tried numerous changes to my input and I always get this error. My primers have degenerate bases in them. Does anyone know if these cause Primer-BLAST problems? Are there any good alternatives out there for checking primer specificity?

Hi,

I need to do HepG2 transfection with Lipofectamine® 3000 from Thermofisher. On the website they say the efficiency is > 80 %.

I am curious whether it can be this high using the standard protocol provided? Is there any optimization to the protocol?

Also, what is the efficiency like if electric transfection is used?

Many thanks,

I am trying to choose between 2 transfection reagents knowing that I will be working with HepG2 cell line and HUH-7

Hello everyone,

Is there anyone working with lipid-based nanoparticles (LPNs) for mRNA delivery who can share their thoughts?

Currently, I am working with PLGA:DOPE:DOTAP nanoparticles, but I am facing significant difficulties in achieving successful transfection in various cell lines (HEPG2, HEK293, JurkatE61). I have already attempted mRNA encapsulation and adsorption, but none of the formulations have yielded any transfection results.

I would greatly appreciate any insights or suggestions on improving the transfection efficiency with LPNs and mRNA delivery in these cell lines.

Thank you in advance for your assistance.

I have been transiently transfecting HepG2 cells with our luciferase reporter alone, or with expression plasmids of interest with the Mirus TransIT 2020 reagent. In the past I have gotten great signal with the reporter alone cells. Now when I try to repeat the experiments I am not getting any luciferase signal from the reporter alone cells, but I am still getting signal from the cells transfected with the luciferase reporter and the expression plasmids. When I check the normalizer values I am getting low values for the luciferase alone cells, but normal ranges for the luciferase and expression plasmids cells. I used an endotoxin removal kit on these plasmids before using them for transfection. Any ideas as to why my luciferase reporter alone cells are not doing so well?

Recently, I have investigated HBV and I wish to make NTCP overexpressed HepG2 which is receptor of HBV. 

To make stably NTCP overexpressed HepG2, I used G418 selection vector, pcDNA3.1(+). Three days after transfection NTCP-FLAG-pcDNA3.1(+) to HepG2, I conducted immunofluorescence and I confirmed about 50% of HepG2 expressed NTCP-FLAG. Since then, I seeded these transfected cells at 0.25 x 10^6 cells/well on 6 well plate. And I used DMEM: 750ug/ml G418, 10% FBS. I changed the media every 3 or 4 days. 

2 weeks later, in case of non-transfected HepG2, there was no growth because of 750ug/ml G418. Unlike this, NTCP-FLAG HepG2 formed several colony, and I transferred it new 6 well plate. 2 weeks later, I conducted immunofluorescnece, however, I confirmed less than 20% of HepG2 expressed NTCP-FLAG. 

In terms of growth, G418 was work well, but, in terms of target protein, NTCP-FLAG, HepG2 was not selected by G418. This is my third attempt. The results of previous first and second experiment were similar. 

What is the problem and is there something I missed?

Is there anyone who have know-how for G418-HepG2 selection? 

Or..is this related to character of NTCP that I didn't know? 

I have been trying to establish a stable cell line using Knockout^TM Single Vector Inducible RNAi System from Clontech. I used Lipofectamine2000 for transfection and then I have selected the cells in 400 micrograms of G418 for 2 weeks. Unfortunately only very less cells survived. When i tried transfection method with different plasmid and it seemed good, however, it was only transient transfection. If anyone tried similar experiment can you give me some advices?

Can anyone share their knowledge in getting synthetic peptides into these or other types of cells?

Thanks!

Hi

I want to transfect HepG2 cells with FGF21 siRNA. Do I have to use all of the Positive control siRNA, Negative control siRNA, Transfection control siRNA, Mock transfection control, Untransfected cells control?