Difference between revisions of "Electroporation of 3T3-L1 Adipocytes"
From Bridges Lab Protocols
Davebridges (Talk | contribs) |
Davebridges (Talk | contribs) (changed default siRNA to 5 uL (500 pmoles) and made some extra notes) |
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==Materials== | ==Materials== | ||
| − | *Differentiated cells FBS day 3 or less. | + | *Differentiated cells FBS day 3 or less. If cells are difficult to trypsinize, they will not recover well. |
| + | **To calculate the number of cells, use 1 - 15cm dish per final dish (10cm/6well/12well). | ||
| + | **For example for 2 knockdowns, each being seeded in 6 - 12 well sized wells, use 1 dish, and 2 electroporations. | ||
*Gene Pulser cuvette | *Gene Pulser cuvette | ||
*DNA: CsCl Purified or siRNA (resuspended to 0.1 nmol/uL with 200 uL water) | *DNA: CsCl Purified or siRNA (resuspended to 0.1 nmol/uL with 200 uL water) | ||
| Line 11: | Line 13: | ||
==Protocol== | ==Protocol== | ||
#Warm media and PBS but not trypsin in water bath | #Warm media and PBS but not trypsin in water bath | ||
| − | #Wash cells twice with PBS (-/-), then trypsinize and with 2 mL 0.25% trypsin in the | + | #Wash cells twice with PBS (-/-), then trypsinize and with 2 mL 0.25% trypsin in the incubator |
#When cells have fallen off the plate, add 9 mL Media and pipet into a 15 mL falcon tube | #When cells have fallen off the plate, add 9 mL Media and pipet into a 15 mL falcon tube | ||
#Spin at 2000 RPM for 5 min. | #Spin at 2000 RPM for 5 min. | ||
#Wash cells with PBS +/+ | #Wash cells with PBS +/+ | ||
#Reuspend cells to a final volume of 0.5 mL per electroporation in PBS +/+ | #Reuspend cells to a final volume of 0.5 mL per electroporation in PBS +/+ | ||
| − | #500 uL of cells is mixed with 50-200 ug of DNA (or | + | #500 uL of cells is mixed with 50-200 ug of DNA (or 5 uL siRNA) in a 0.4 cm cuvette. These volumes are for half of a final plate of cells. Scale up or down accordingly. |
| − | #Electroporate at | + | #Electroporate at 160V and 950 uF |
| − | #Add 1 mL media to cuvette and transfer to a 15 mL tube with media for 10 min. | + | #Add 1 mL media to cuvette and transfer to a 15 mL tube with media for 10 min. The amount of media should correspond to the final volume of plated cells (ie one electroporation of half a plate of cells would go into 6 mL final volume of media). |
#Aspirate floating debris before replating | #Aspirate floating debris before replating | ||
#Bring up tube to final required volume, mix and plate | #Bring up tube to final required volume, mix and plate | ||
Latest revision as of 18:45, 17 February 2010
Materials
- Differentiated cells FBS day 3 or less. If cells are difficult to trypsinize, they will not recover well.
- To calculate the number of cells, use 1 - 15cm dish per final dish (10cm/6well/12well).
- For example for 2 knockdowns, each being seeded in 6 - 12 well sized wells, use 1 dish, and 2 electroporations.
- Gene Pulser cuvette
- DNA: CsCl Purified or siRNA (resuspended to 0.1 nmol/uL with 200 uL water)
- PBS +/+
- PBS -/-
- 0.25% Trypsin
- L1 FBS Media
Protocol
- Warm media and PBS but not trypsin in water bath
- Wash cells twice with PBS (-/-), then trypsinize and with 2 mL 0.25% trypsin in the incubator
- When cells have fallen off the plate, add 9 mL Media and pipet into a 15 mL falcon tube
- Spin at 2000 RPM for 5 min.
- Wash cells with PBS +/+
- Reuspend cells to a final volume of 0.5 mL per electroporation in PBS +/+
- 500 uL of cells is mixed with 50-200 ug of DNA (or 5 uL siRNA) in a 0.4 cm cuvette. These volumes are for half of a final plate of cells. Scale up or down accordingly.
- Electroporate at 160V and 950 uF
- Add 1 mL media to cuvette and transfer to a 15 mL tube with media for 10 min. The amount of media should correspond to the final volume of plated cells (ie one electroporation of half a plate of cells would go into 6 mL final volume of media).
- Aspirate floating debris before replating
- Bring up tube to final required volume, mix and plate
