Team:Debrecen-Hungary/protocols/ Transfection
From 2010.igem.org
Transfection protocol with PEI nuclear receptor construct (for COS1 cells)
Scientific BackgroundThe following protocol is used to succesfully introduce foreign plasmid DNA into pluripotent cells through a chemical way, PEI mediated transfection. PEI stands for Poliethilenimine, a cationic polymer which can bind nucleic acids and helps the DNA to get into the cells by receptor-mediated endocytosis. OverviewPerforming the protocol from beginning to end takes approximately one and a half day and it is divided into two major procedures: 1.Plating COS1 cells into 48 well plates in order to get 60% confluency of adherent cells for transfection The goal is to get 60% confluency of adhaerent cells on a 48 well plate before transfect them 2. The PEI mediated transfection itself The goal is to introduce foreign plasmid DNA successfully into COS1 cells 24 hours after the plating MaterialsI.Plating COS1 cells:48 well plates DMEM Medium containing 10% FBS Trypsin-EDTA 1x PBS Brüker-chamber centrifuge tubes serological pipettes pipettes and tips vacuum aspirator Pasteur pipettes Sterile laminar air flow box incubators
II. PEI mediated transfectionPlated cells Serum Free DMEM Medium sterile 100 mM PEI solution sterile 150 mM NaCl solution TE –buffer (filtered) Plasmids: Beta-Gal (normalizer), Luciferase (tracer), Receptor1, Receptor2, VDR-1 (MOCK, negative controll) - their concentration has been measured previously Eppendorf tubes centrifuge tubes serological pipettes pipettes and tips vacuum aspirator Pasteur pipettes Sterile laminar air flow box incubators
ProcedureI. Plating COS1 cells:1. Prepare the sterile box, and prewarm the medium, trypsin-EDTA and PBS up to 37°C 2. Get the cells to the sterile box in a closed cell culturing flask or Petri dish. 3. Gently remove used medium from the cells using vacuum aspirator with Pasteur pipette 4. Wash the cells with 2-3 ml PBS, then remove it gently by vacuum aspirator with Pasteur pipette 5. Incubate the Cos1 cells for 3-5 mins with 2 ml of trypsin-EDTA at 370C, 6. Add 4 ml medium(you can change the dilution level depending on the cell number, in order to be able to count the cells easier) to the trypsinised cells, 7. Prepare the Bürker-chamber and do a cell count 8.To reach 60% confluency the day after plating, we put 21.000 cells into each well For a 48 well plate, if we calculate with 60 wells, we put into a 50 ml centrifuge tube:
- 21000x60= 1.320.000 cells [ cellnumber in 1 ml / 1.320.000 ML cell susp. ] , and if the total volume of the wells are 200 ul,
- we fill the cell suspension up to 200x60 ul= 12 ml with 10% FBS DMEM 9. By using a repeating pipet, put 200 ul from this suspension into each well, swirl the plate 10. Incubate the cells for 1 day at 37°C, 5% CO2
II.PEI mediated transfection1.Prepare the sterile box, and prewarm the medium up to 37°C 2.Change the medium at least 1 hour before transfection to Serum Free DMEM. 3. Mix gently plasmid solutions (Do not vortex!) 4. Dilute plasmids to 0,1 µg/µl cc. with TE-buffer in sterile Eppendorf tubes 5. Prepare DNA mixes: T A B L E I N S E R T R E Q U I R E D Put 66-66 ul Mastermix 1 into four Eppendorf tubes. The plasmids of special receptors have to be added to each Eppendorf tubes (7 µl from each) in the case of PPAR gamma construct transfection: 6. Prepare PEI mix in 4 pieces of eppendorf tubes: A. Per 1 well: B. Per 16 well: vortex the PEI- NaCl mixes in the Eppendorf tubes briefly.
7. Add PEI mixes to DNA mixes dopwise , then vortex briefly. (Do not add DNA mixes to PEI mix!) 8. Incubate transfection mixes for 20 mins at room temperature 9. Add total transf.mix.volume in one epp.tube/16 µl transfection mix to each well in drops, then swirl gently the plates 48-well plates, 8 rows, 6 columns: 1st row: (VDR-)x2 2nd row: Rec1+ VDR- 3rd row: Rec2+VDR- 4th row: Rec1+Rec2 5th row:(VDR-)x2 6th row:Rec1+ VDR- 7th row:Rec2+VDR- 8th row:Rec1+Rec2
Notes & troubleshootingReferences1. Horbinski C, Stachowiak MK, Higgins D, Finnegan SG. Polyethyleneimine-mediated transfection of cultured postmitotic neurons from rat sympathetic ganglia and adult human retina. .[http://www.biomedcentral.com/1471-2202/2/2 BMC Neurosci. 2001;2:2]. 2. Pollard H, Remy JS, Loussouarn G, Demolombe S, Behr JP, Escande D: Polyethylenimine but not cationic lipids promotes transgene delivery to the nucleus in mammalian cells.[http://www.jbc.org/content/273/13/7507.long J Biol Chem 1998], 273:7507-7511 Links[http://www.youtube.com/user/debrecenigem2010#p/u/13/r8DlyOCRJRs Video I] [http://www.youtube.com/user/debrecenigem2010#p/u/12/IgRxD-Tf5Eo Video II] |