Difference between revisions of "Transfecting Cells"
From SDMRC
| (2 intermediate revisions by the same user not shown) | |||
| Line 1: | Line 1: | ||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
==Summary== | ==Summary== | ||
Cos 1 (African green monkey, from kidney cells). Cells are heterozygous, they all look different. We are transfecting Cos 1, 6 cm plates | Cos 1 (African green monkey, from kidney cells). Cells are heterozygous, they all look different. We are transfecting Cos 1, 6 cm plates | ||
| Line 21: | 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 | + | #Take sterile tubes for as many transfections you have, label (in this example we needed six tubes) |
| − | + | #Put 150 ul of media into tubes | |
| − | Put 150 ul of media into tubes | + | (You can reuse the tip if you don’t touch anything) |
| − | 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 |
| − | 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) |
| − | Into the 150 ul of media add about 1-2 ug of DNA (optimize DNA concentration first) | + | # Tap gently to mix |
| − | Tap gently to mix | + | # Add 6.5 ul of lipofectamine (transfection media) |
| − | Add 6.5 ul of lipofectamine (transfection media) | + | # Pipette directly in the tube, flush, and tap |
| − | Pipette directly in the tube, flush, and tap | + | # re-cap / close |
| − | re-cap / close | + | # Let it sit for 5-10 minutes (helps enhance transfection efficiency) |
| − | 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) |
| − | 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 |
| − | 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 |
| − | 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
