WangLab:Transfection: Difference between revisions
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(New page: === Procedures === Lipofectamine 2000 method (Invitrogen) (for 1 well in 6-well cluster 10cm2, small dish of 35 mm). # The day before transfection, pass confluent cell 1:3 in 10%FBS withou...) |
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=== Description === | |||
=== Materials === | |||
=== Total time === | |||
6.5 hours | |||
=== Procedures === | === Procedures === | ||
Lipofectamine 2000 method (Invitrogen) (for 1 well in 6-well cluster 10cm2, small dish of 35 mm). | Lipofectamine 2000 method (Invitrogen) (for 1 well in 6-well cluster 10cm2, small dish of 35 mm). | ||
| Line 5: | Line 9: | ||
# 1 ug DNA gently mixed into 100 ul Optimum only, gently tap the tip of vial to mix. | # 1 ug DNA gently mixed into 100 ul Optimum only, gently tap the tip of vial to mix. | ||
# 2 ul lipofectamine mixed into 100 ul Optimum only, gently tap the tip of vial to mix. | # 2 ul lipofectamine mixed into 100 ul Optimum only, gently tap the tip of vial to mix. | ||
# Wait for 5 min, gently apply lipofectamine-Optimum to DNA-Optimum. Tilt and rotate the DNA tube, while adding lipo-optimum drop by drop on | # Wait for 5 min, gently apply lipofectamine-Optimum to DNA-Optimum. Tilt and rotate the DNA tube, while adding lipo-optimum drop by drop on its wall. | ||
# Tap or reverse the tube gently to mix. | # Tap or reverse the tube gently to mix. | ||
# Incubate in RT for 20 min for the complex formation between DNA and lipofectamine | # Incubate in RT for 20 min for the complex formation between DNA and lipofectamine | ||
# Apply the DNA-Lipofectamine complex Optimum to cells. Gently swirl the dish to mix. | # Apply the DNA-Lipofectamine complex Optimum to cells. Gently swirl the dish to mix. | ||
# Incubate 5.5 hr in incubator. check cell toxicity, if severe, stop the incubation and apply fresh 0.5% FBS-DMEM, if not, continue incubation until next day and change to 0.5%FBS DMEM. | # Incubate 5.5 hr in incubator. check cell toxicity, if severe, stop the incubation and apply fresh 0.5% FBS-DMEM, if not, continue incubation until next day and change to 0.5%FBS DMEM. | ||
=== Notes === | === Notes === | ||
Latest revision as of 09:15, 27 August 2007
Description
Materials
Total time
6.5 hours
Procedures
Lipofectamine 2000 method (Invitrogen) (for 1 well in 6-well cluster 10cm2, small dish of 35 mm).
- The day before transfection, pass confluent cell 1:3 in 10%FBS without Penicillin/Straptomyosin.
- Check the cell condition, if it is in 60-80% confluency, proceed, otherwise, start a new experiment.
- 1 ug DNA gently mixed into 100 ul Optimum only, gently tap the tip of vial to mix.
- 2 ul lipofectamine mixed into 100 ul Optimum only, gently tap the tip of vial to mix.
- Wait for 5 min, gently apply lipofectamine-Optimum to DNA-Optimum. Tilt and rotate the DNA tube, while adding lipo-optimum drop by drop on its wall.
- Tap or reverse the tube gently to mix.
- Incubate in RT for 20 min for the complex formation between DNA and lipofectamine
- Apply the DNA-Lipofectamine complex Optimum to cells. Gently swirl the dish to mix.
- Incubate 5.5 hr in incubator. check cell toxicity, if severe, stop the incubation and apply fresh 0.5% FBS-DMEM, if not, continue incubation until next day and change to 0.5%FBS DMEM.
Notes
- The optimal time interval between passing cells and transfection is 15-18 hours; and that between transfection and imaging is 36-54 hours.
