Pelling:Protocols/Troubleshooting Transfections

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Transfection procedures are highly dependent on the cell type and plasmid being used. Below are some alternative methods people have had success with in the past. Alternative Method 1 Person Zeinab Al-Rekabi Cell Line C2C12 Rat Myoblast Plasmids which work well Zyxin-RFP Plasmids which do not work well Vinculin-GFP The day before transfections, I seed my cells to 90-100% confluency in 100cm plate. Next day Follow the same protocol as Invitrogen split cells Use a dilution of 1:4 (works best in my case) Instead of adding optimem and incubating for 4-6hrs, I simply add the growth media and I find better and much stronger expression than using optimem. 24 hours after transfection, change media check with fluorescence 48 hours after transfection, check under fluorescence and I generally get better expression and from there further experiments and imaging as AFM/confocal, AFM/EPI, or staining can be done. Alternative Method 2 Person Tina Haase Cell Line HeLa and MDCK Plasmids which work well PLC-δ One day prior to doing transfections, I split my cells normally (~70% confluency in 100cm plate) and add ~50µl (from pellet re-suspended in 2 ml of media) to each of my 35 ml glass dishes. The following day Use the Invitrogen protocol (but skip the step where you split with Optimem) Use dilution of 1:2 (so 2µl of LF2K in 48µl of Optimem) Usually I get a concentration between 300-500 µg/ml of PLC-δ plasmid DNA, which corresponds to ~3µl in 47µl Optimum, or ~2µl in 48µl Optimem, respectively After combining the LF2K/DNA/Optimem (your 100µl solution) let it sit for at least 10 minutes, and discard the normal media from your 35 ml dishes and add ~1ml of Optimem There is no need to wait the extra hour, just add your DNA to the dishes and incubate for 4-5 hours. I have tried skipping the 4-6 hour incubation period and although you do get some fluorescing cells, not nearly as many as when you have some patience…which will give you upwards of 50% of your dish fluorescing! Add excess media (I try to squeeze in 2 ml) following the 4-6 hour incubation period. Change the media the following day. Expression is already really good on the first day! 48 hours after doing transfections the cells are usually still glowing brightly and should be a complete monolayer (actually I had them fluoresce up to 5 days later…but you will have a lot of cells…way more than necessary) Alternative Method 3 Person Pato Kunda Cell Line HeLa and RPE1 Plasmids which work well none in particular; it describes a general procedure of transfection with Fugene Day 1: Plate cells the day before, so they will be at around 80% confluency Day 2: Change media with PenStrep free DMEM In a 1.5 ml microcentrifuge tube, make up plasmid DNA solution in optiMEM (see table below) Add appropriate volume of Fugene (see table below) Incubate at room temperature for 20 minutes Pipette solution over the cells and return to 37 deg. Day 4: transient transfection will be at maximum HeLa cells Use 3:1 ratio for large volumes; 6:1 for smaller volumes (μL Fugene:μL plasmid) Total vol. Vol. OptiMEM Amount DNA Vol. Fugene 10cm plate 10ml 500μl 10μg 30μl 12-well plate 1ml 50μl 500ng 1.5μl 4-well chamber slide 500μl 10μl 200ng 1.2μl (6:1) 8-well chamber slide 100μl 5μl 100ng 0.6μl (6:1) RPE1 cells (retinal pigment epithelial) Use 6:1 ratio for large volumes; 6:1 for smaller volumes (μl Fugene:μl plasmid) Use DMEM/F12 Total vol. Vol. OptiMEM Amount DNA Vol. Fugene 10cm plate 10ml 500μl 10μg 60μl 12-well plate 1ml 50μl 500ng 3μl 4-well chamber slide 500μl 10μl 200ng 1.2μl 8-well chamber slide 100μl 5μl 100ng 0.6μl