Difference between revisions of "Transfecting Cells"
From SDMRC
(Created page with "==Summary== Cos 1 (African green monkey, from kidney cells). Cells are heterozygous, they all look different. We are transfecting Cos 1, 6 cm plates * Make sure everything is...") |
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===Materials=== | ===Materials=== | ||
| − | *DNA, transfection lipofectamine 2000 | + | * DNA, transfection lipofectamine 2000 |
* Antibiotic free DMEM (no serum), kept in the fridge | * Antibiotic free DMEM (no serum), kept in the fridge | ||
* Sterile tips, tubes, pipettes (sprayed) | * Sterile tips, tubes, pipettes (sprayed) | ||
| Line 10: | Line 10: | ||
* Work deep inside the hood | * Work deep inside the hood | ||
| − | + | ===Transfection mix (for this example)=== | |
| − | ==Transfection mix (for this example)== | + | #Take sterile tubes for as many transfections you have, label (in this example we needed six tubes) |
| − | + | #Put 150 ul of media into tubes | |
| − | + | (You can reuse the tip if you don’t touch anything) | |
| − | + | # Make sure you don’t go over any open tubes or bottles, close tubes and bottles right away | |
| − | + | # Into the 150 ul of media add about 1-2 ug of DNA (optimize DNA concentration first) | |
| − | + | # Tap gently to mix | |
| − | + | # Add 6.5 ul of lipofectamine (transfection media) | |
| − | + | # Pipette directly in the tube, flush, and tap | |
| − | + | # re-cap / close | |
| − | + | # Let it sit for 5-10 minutes (helps enhance transfection efficiency) | |
| − | + | # After optional 5-10 minutes, grab cell plates from incubator (careful not to open lid yet) | |
| − | + | # Make sure tubes and plates are in order (1-6), set pipette to 170 (ish) ul, take up media from tube and add to plate (pipette at an angle so nothing falls in) spread media in drops in a circle | |
| − | + | # Give the dishes a gentle swirl, and place in incubator for a minimum of 24 hours and a maximum of three days. After 24 hours the DNa will be taken up, but after three days they will over grow and die | |
| − | + | ||
| − | + | ||
Latest revision as of 21:30, 20 July 2018
Summary
Cos 1 (African green monkey, from kidney cells). Cells are heterozygous, they all look different. We are transfecting Cos 1, 6 cm plates
- Make sure everything is clean and sterile
Materials
- DNA, transfection lipofectamine 2000
- Antibiotic free DMEM (no serum), kept in the fridge
- Sterile tips, tubes, pipettes (sprayed)
- Make sure cells are 50-70% confluent
- Work deep inside the hood
Transfection mix (for this example)
- Take sterile tubes for as many transfections you have, label (in this example we needed six tubes)
- Put 150 ul of media into tubes
(You can reuse the tip if you don’t touch anything)
- Make sure you don’t go over any open tubes or bottles, close tubes and bottles right away
- Into the 150 ul of media add about 1-2 ug of DNA (optimize DNA concentration first)
- Tap gently to mix
- Add 6.5 ul of lipofectamine (transfection media)
- Pipette directly in the tube, flush, and tap
- re-cap / close
- Let it sit for 5-10 minutes (helps enhance transfection efficiency)
- After optional 5-10 minutes, grab cell plates from incubator (careful not to open lid yet)
- Make sure tubes and plates are in order (1-6), set pipette to 170 (ish) ul, take up media from tube and add to plate (pipette at an angle so nothing falls in) spread media in drops in a circle
- Give the dishes a gentle swirl, and place in incubator for a minimum of 24 hours and a maximum of three days. After 24 hours the DNa will be taken up, but after three days they will over grow and die
