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Position: Discussion forums > Bioscience biotechniques discussion forums > Molecular biology troubleshooting forum
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Transfection
Posted by: EANON (IP Hidden, Junior member, 19)
Date: July 11, 2005 09:33AM
I am transfecting a GFP-tagged protein into HEK293 cells using the Lipofectamine 2000 reagent. When I check with a fluorescence microscope, my yield is low (about 10-15% vs 70% typical). When I harvest cells and check with Western, my protein is not there at all.
Did anybody have a similar problem? I am following the manufacturer's instructions (add lipofectamine in Optimem media + DNA in optimem for ~20 minutes, then putting onto cells, then change media after 4-6 hours, and harvest next day). My DNA conc is ~400-600 ug/ml.
Re: Transfection
Posted by: aniu1229 (IP Hidden, New member, 8)
Date: July 11, 2005 10:37AM
It is abnormal. I also did similar experiments, but the efficiency is always high. I used LF2000 before, and now superfect from QIAGEN. I feel both are good, just superfect has less toxicity, which might be better for 293, I think. as 293 cells are very fragile.
u can transfect empty GFP vector as a control, to see whether there is something special with ur protein. and when u use LF2000, I suggest high confluency of cells, higher than 90%, due to the toxicity of LF2000. by the way, u mentioned the concentration of ur DNA, but how much did u add to cells? I remembered that in order to get good efficiency, I did add a lot of DNA and LF2000. in my case, the best ratio between them is 1:3. and I use up to 20 microgram DNA for cells in 90mm dish. it is really a lot. but if u use 10 microgram for this dish, efficiency should also be ok. if it is ur first time to do this transfection, then it is better to optimize ur own condition as written in the manual. good luck!
Re: Transfection
Posted by: thomas. (IP Hidden, Unregistered user, )
Date: July 12, 2005 09:35AM
Sometime lipo2000 causes problem in addition to toxicity for very common cell lines, you are not alone especially the sensitive, easy to detach 293 (the easist and the worst cells to work with).
Your protocol seems all right to me. All I can think of is may be DNA purity, cell density issues? I don't think increase DNA concentration helps (lipo2000 need to increase proportionally too and that is not good), stick to the instruction. If it is still not working, try another reagent. Our lab has changed to Gencarrier-1 from lipo2000/fugene because it is more consistent, effective and much cheaper. lipo2000 is not consistent and has toxicity issue when longer exposure and fugene is well too expensive.
Re: Transfection
Posted by: EANON (IP Hidden, Junior member, 19)
Date: July 14, 2005 10:25AM
Thanks for your advice. Because I did not have empty GFP vector immediately available, I transfected another GFP-protein as control and sure enough, that protein transfected nicely (>60%), while my protein of interest did not (~15%). This protein has been transfected before, so the problem is my source of plasmid rather than the protein itself.
After I ran ~25 ng of the whole plasmid on a 1% agarose gel, I observed some smearing. I assumed this was due to contamination by bacterial genome, so I transformed into E. Coli cells and extracted DNA using Maxiprep. My measured DNA conc appeared to be pure when measured by 260/280 and at good concentration. However, this DNA also did not give good transfection efficiency and I am sure when I run the agarose gel I'll get similar results as before. Does anybody know how I can purify good plasmid free from contamination and without smearing?
plasmid maxiprep
Posted by: ad (IP Hidden, Unregistered user, )
Date: August 18, 2005 10:13PM
i did a plasmid maxiprep by the alkaline lysis method. everything was done according to maniatis protocol. but int he last step i did not allow sufficient time for the pellet to dry after 70% ethanol wash. then when i added TE to the pellet, the whol thing turned white. the nucleic acid pellet when dissolved should be colourless. but there is a whitish precipitate and solution is not clear. is there any way i can rectify this damage?
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