| |||||||||
| Login :: Register :: Search forums :: Top Users :: Reagent |
|
Position: Discussion forums > Bioscience biotechniques discussion forums > Cell biology and histology methods forum
|
NIH3T3
Posted by: Stanford (IP Hidden, New member, 1)
Date: June 12, 2005 02:14PM
Has anyone used this commercial reagent called Transfectin Lipid Reagent from Bio-Rad? I've been trying to transfect NIH 3T3 cells with an egfp - tagged plasmid and used all the options they suggst in terms of dna-transfectin ratio as well as increasing amount of dna. max i've used so far is 2ug. i still get extremely poor transfection efficiency. i'm new to this technique and would appreciate advice / suggestions from people who've done this or worked with this cell line before!
thanks
Re: NIH3T3
Posted by: 3'-UTR (IP Hidden, Unregistered user, )
Date: June 12, 2005 02:23PM
1. Did you check if they (Bio-rad) have carried out transfections on your cell line. Usually they provide a list of cell lines that they have tested their product on.
2. Did you check the quality of your plasmid. Make sure the absorbance ratio is >1.8. Check the confluence of your cells. 3. If you definitely want to use only the Bio-rad product then use the other transfection agent just as a positive control.
Re: NIH3T3
Posted by: jurkatmom (IP Hidden, Unregistered user, )
Date: June 12, 2005 02:30PM
NIH 3T3 is a very commonly used cell line. We have lots of people switch to a new transfection reagent called Gencarrier-1 here at CSHL (Cold Spring Harbor Laboratory). It outperforms many other lipo reagents like lipofectamine2000, fugene-6, GenPORTER, Genejuice, jetPET, effectene, Superfectene......in efficiency, low toxicity, consistency and cost. Our experience is if Gencarrier can not do the job, switch to other methods - electroporation, nucleofection or use adeno/retroviral system, and don't waste yout time and money on trying the other lipo reagents.
Re: NIH3T3
Posted by: mcmv (IP Hidden, New member, 1)
Date: June 14, 2005 07:42AM
I have had reasonably good success with using Transfectin for NIH3T3 cells. I use a ratio of 2 ul Transfectin per ug DNA. Some factors which may help. Transfection with this reagent works better when cells are seeded at a higher density than is optimal for many other transfection procedures. The cells should be seeded 24 hours before transfection. Make sure to do you DNA/reagent mixes in polystyrene rather than polypropylene tubes. Once the DNA mix is added to the cells leave it on for 24 hours, there is little cytotoxicity and the efficiency is better. One major problem with this transfection reagent- it is great for transfecting single plasmids but in cotransfections there is a high incidence of cells getting only one or the other plasmid, not both. Both our lab and another lab found the same thing with several cell lines with this reagent, independently.
Re: NIH3T3
Posted by: thomas (IP Hidden, New member, 5)
Date: July 3, 2005 01:19PM
the ONLY reagent that worked for NIH3T3 for us was Lipofectamine
|
We are moving ... Please post to our new community forums
|
|
||||||||