Hello everybody, I am facing some major problems in transfecting a GFP::Protein in HeLa K cells. Briefly I am using a plasmid for transient expression in human cells, this plasmid has a pSV40 promoter. The total size of the plasmid is around 7,5 Kb. I transfect the cells using XtremeGene-9 and I look at the fluorescence after 48 hours. The problem is that very few cells are green, no more than 1-2%, and I cannot work with such a bad efficiency. I have altready tried to increase the amount of DNA and the ratio between the DNA and the transfection reagent. I have also tried to change cell density but the results do not change much. Any advice?
Plasmids, when transfected into HeLa, need to be compacted for entry. GFP can easily be transfected, but be sure to use a complex condenser (https://altogen.com/product/hela-transfection-reagent-cervix-adenocarcinoma-cells/) and then monitor for cell vitality to see if you need to alter the concentration of reagent. I agree with the rest that you need to review the experimental set up.
Do you know that your plasmid works? By that I mean, do you know that if you express your plasmid in cells, you can see GFP? It looks like you might have a GFP:fusion protein. Fusing two proteins together can often mess them up.
I would suggest trying to transfect another cell type, or at the very least, get your hands on a GFP plasmid that you KNOW works, and use that as a positive control.
An alternative transfection reagent might also be helpful.
Good luck,
J
Thank you all!
Since the most common advice is to change the reagent I will try this way. I will let you know!
you might also try the calcium phosphate method; it works fine in our hands with HeLa
Hi Luca,
yes your plasmid can be a problem, to answer this question you can use a positive control meaning a transfection grave plasmid-GFP
http://www.ozbiosciences.com/transfection-controls/55-plasmids-pvectoz.html
and also try another transfection reagent such as DreamFect or DreamFect Gold because it has been very successful in transfecting HeLa cells as demonstrated by the publications
http://www.ozbiosciences.com/transfection-dna/19-dreamfect-gold-transfection-reagent.html
here are few publications:
http://www.ncbi.nlm.nih.gov/pubmed/21857679
http://www.ncbi.nlm.nih.gov/pubmed/20724658
http://www.ncbi.nlm.nih.gov/pubmed/25148142
http://www.ncbi.nlm.nih.gov/pubmed/25116916
If you want samples of DreamFect and DreamFect Gold let me know at [email protected]
Good Luck
Olivier
HI.
Most of the answers above are good ones. I would add that you should also check to make sure you DNA is good quality. Linearize your plasmid by digestion and make sure it runs as a single band instead of a smear. Make sure the intensity of the band is appropriate for the amount you load.
Additionally, you can transfect your plasmid into HEK 293 cells. These will take up most anything using almost any transfection reagent. This may help determine whether your problem lies with the plasmid or the cells.
Finally, find a positive control. Something with GFP that has worked recently in someone else's hands.
Dear Luca, As I have undergone the same transfection problems in my earlier experimentation, Here are some of the points to make clear and tackle the problems.
1. The quality of plasmid is very crucial for transfection, especially what is the elution buffer in the final step and in which plasmid is kept. The presence of ethanol can be lethal to the cell and hamper transfection.
2. The confluency of cell in which tranfection is performed. If you want to perform the experiment for 3-4 days, make the confluency, slight lower. Generally, 50-60% confluent growth is fine.
3. The quality of tranfecting agent. In similar fashio for tranfection of 9kb egf tagged HIV virus, I used lipofectamine 2000 in HEK and HeLa cell lines, which proved good in this reagard.
4. The tranfecting protocol is also crucial. The handling and incubation should be monitored.
Hope this helps you. Thanks.
- Badges
- Science topic
- Similar topics
- Methodology
- Laboratory
- Cellular Biology Techniques