Materials:

	Lipofectamine
	IMDMcontaining10%fetalbovineserum,1%glutamine,1%aa
	IMDMcontaining1%glutamine
	IMDMcontaining20%fetalbovineserum,1%glutamine,1%aa

1. Inasix-wellor35mmtissuecultureplate,seed~2x10⁵cellsperwellin2mlIMDMcontaining10%FBSandnonessentialaminoacids.
2. Incubatethecellsat37ECinaCO₂incubatoruntilthecellsare70-80%confluent.Thiswillusuallytake18-24h.
3. Preparethefollowingsolutionsin12x75mmsteriletubes:SolutionA:Foreachtransfection,dilute2μgDNA(plasmid)in375μlserum-freeIMDM(containingnonessentialaminoacids).SolutionB:Foreachtransfection,dilute12μlLIPOFECTAMINEReagentin375μlserum-freeIMDM.
4. Combinethetwosolutions,mixgently,andincubateatroomtemperaturefor15-45min.Thesolutionmayappearcloudy,howeverthiswillnotimpedethetransfection.
5. Washthecellsoncewith2mlserum-freeIMDM.
6. Foreachtransfection,add750μlserum-freeIMDMtoeachtubecontainingthelipid-DNAcomplexes.Donotaddantibacterialagentstomediaduringtransfection.Mixgentlyandoverlaythedilutedcomplexsolutionontothewashedcells.
7. Incubatethecellsfor5hat37ECinaCO₂incubator.
8. Add1.5mlIMDMwith20%FBSwithoutremovingthetransfectionmixture.Iftoxicityisaproblem,removethetransfectionmixtureandreplacewithnormalgrowthmedium.
9. Replacemediumat18-24hfollowingstartoftransfection.
10. Assaycellextractsforgeneactivity24-72hafterthestartoftransfection,dependingoncelltypeandpromoteractivity.

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