You may try HEK293T cells. I dont quite understand what you mean by gene is confused with HA tag. It would be better if you check if your construct is in frame, and if you get expression of the positive control of some other protein recombined in the same way

@Mahesh. Thansk very much for your response. Sorry for the misspelling. I mean the protein fused with a HA tag. And i do checked the construction, it is good. And i also had a positive control, which worked all the time.

Hi, Mingxiang, I would like to add some comments below:

1. First you need to make sure that the expression plasmid is truely delivered into the HEK293 cells. sometimes the transfection efficiency dramatically goes down if using a bigger vector construct. I would like to recommend PEI as trasfection reagent in place of Lipofectamin3000. I have more than 10-year-experience by using PEI to transfect 293T and it never let me down even my plasmid is more than 10K bps.

2. How did you check the expression of your target gene? by Western Blot? or some other techniques? You may need to look into the QC assay and see if there is any room to improve.

Good luck

Gary

@Gary Jing.

Thanks for your response. Do you have a detailed proctocol for that. And do mind to share what kind of product you used?

Thanks

Hi Zhang,

Few thoughts

- Use ncbi orf finder , and check about the stop codons. 

- Check if  your start codon is further away from the rbs.

- Are you using codon optimised gene?, this will increase expression significantly. 

- Do you have 3' UTR sequence in your construct ? Kozak will increase protein expression.

- How is your cell growth as compared to the normal positive control, are they healthy? Most important is to figure out if your gene is toxic to the cell.

- You can also try transfection using media which dosent contain antibiotics. 

@Mahesh, thanks for your response.

Regarding your third coments, how do you modified codon?

We purchase the PEI powder from Polyscience, the link to this product is http://www.polysciences.com/default/polyethylenimine-linear-mw25000.

Do not disolve this powder by heating, you can first adjust the pH of Saline buffer to 4 and add the PEI powder into it; after disolving, adjust the pH to 7.4 using NaOH, then sterilize through a 0.22um filter.

When you do transfection, keep the ratio of PEI to DNA plasmid at 3:1. Generally the serum in the culture medium does not interfer with the transfection efficiency, so, it's not necessary to use serum-free medium. Remember to refresh the culture medium at 6-8 hours after transfection. You can harvest the cell sample for QC after 48 hours.

Good luck

I think it is better to figure out most of the things which might go wrong. So, I listed couple of important ones to check intensively. Here is explanation of my third comment - Logic is different host exhibit bias towards codons and prefer some codons as compared to others, some species even completely avoid some codons. Optimization is quite easy, you just insert your sequence in various tools and specify your choice.

Here are some :

https://www.genscript.com/tools/rare-codon-analysis

https://www.thermofisher.com/us/en/home/life-science/cloning/gene-synthesis/geneart-gene-synthesis/geneoptimizer.html

http://gcua.schoedl.de/index.html

@ Ted.

Thanks for your response. I had another gene which is around 500bp and has same construction. It works all the time. Can it be considered as a positive control? If you have more comments, please let me know

@Mahesh, thanks for your comments, after i check the usage, how i can modify technically. I mean it’s a bog gene, i can’t synthesize it. Do you have recommendation on that?

Best wishes,

Ming

@Gray.

Thanks for the information

Hi Ming,

Best is to google and find lab which can provide you optmized gene or else you have to place order to the company.

Mahesh Chandak thanks