Lipofectamine-Plus® pcDNA Transfection of Vero Cells or A549 Cells
Lipofectamine-Plus® pcDNA Transfection of Vero Cells or A549 Cells
Lipofectamine-Plus® pcDNA Transfection of Vero Cells or A549 Cells
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The University <strong>of</strong>Ge<strong>or</strong>giaRalph Tripp LabProtocolsSTANDARD OPERATINGPROCEDURESTitle: <strong>Lip<strong>of</strong>ectamine</strong>-Plus ® <strong>pcDNA</strong> <strong>Transfection</strong> <strong>of</strong> <strong>Vero</strong><strong>Cells</strong> <strong>or</strong> <strong>A549</strong> <strong>Cells</strong>No: RTLP-siRNA-1Location:Approval Date: Supersedes Date:Old CCRC Tripp Lab10 September 2004Materials: Page 1 <strong>of</strong> 2•Lab coat•Gloves•Prepared <strong>Vero</strong> <strong>or</strong> <strong>A549</strong>cells•<strong>Lip<strong>of</strong>ectamine</strong>-Plus<strong>pcDNA</strong>•Phosphate BufferedSaline (PBS)•DMEM•Fetal Bovine Serum(FBS)•T-75 <strong>or</strong> T-150flasks•37°C, 5% CO 2incubat<strong>or</strong>•Pipettes•Pipetteman•Pipette Aid•Pipetteman tipsProcedure:Day 1: (Thursday)1. 24h pri<strong>or</strong> to transfection, plate cells in flasks so that they are 50-80% confluentand in log phase.2. Use Dulbecco's Modified Eagles Medium & 10% FBS (DME)Day 2: (Friday)1. Pre-complex the DNA with the "Plus Component":a) Dilute DNA to 1 µg/ul in Serum Free-DME (SF-DME) and mix.b) Th<strong>or</strong>oughly ix the Plus reagent bef<strong>or</strong>e using.c) Add 200 µl <strong>of</strong> DNA to 600 µl <strong>of</strong> Plus reagent and mix.d) Incubate f<strong>or</strong> 15 minutes at room temperature.2. Dilute <strong>Lip<strong>of</strong>ectamine</strong>: 9.6 ml <strong>of</strong> SF-DME + 0.4 ml <strong>of</strong> <strong>Lip<strong>of</strong>ectamine</strong>3. Combine Plus reagent-DNA with diluted <strong>Lip<strong>of</strong>ectamine</strong>, mix well, andincubate 15 minutes at room temperature.4. Decant media from flasks and wash 1x with SF-DME.5. Add back 2.5 ml <strong>of</strong> SF-media/75 cm 2 flask, <strong>or</strong> 5.0 ml <strong>of</strong> SF-DME/150 cm 2flask.
<strong>Lip<strong>of</strong>ectamine</strong>-Plus ® <strong>pcDNA</strong> <strong>Transfection</strong> <strong>of</strong> <strong>Vero</strong> <strong>Cells</strong> <strong>or</strong> <strong>A549</strong> <strong>Cells</strong>Page 2 <strong>of</strong> 26. Add 2.5 ml <strong>of</strong> DNA-<strong>Lip<strong>of</strong>ectamine</strong> complex/well f<strong>or</strong> 75 cm 2 flasks <strong>or</strong> 5.0ml/150 cm 2 .7. Incubate f<strong>or</strong> 3 hours at 37 o C8. Following the 3 hour incubation, add 5 ml <strong>of</strong> DME-20% (2x FCS) to the 75cm 2 flasks <strong>or</strong> 10 ml to the 150 cm 2 flasks.Day 5: (Monday)Give flask to Les f<strong>or</strong> purification <strong>or</strong> freeze in –70 o C
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