930
edits
Changes
Wrote initial protocol for PEI transfections
This protocol is modified from the [https://www.addgene.org/protocols/lentivirus-production/ Addgene website] protocol
== Materials==
* Cells to transfect, generally growing in log phase at ~90% confluence
* Plasmids to transfect. Generally use 500 ng/well of a 6 well plate
* OptiMEM
* Chloroquine stocks (25 mM stocks). See [[Preparation of Chloroquine Stocks]].
* PEI (1 mg/mL stocks). See [[Preparation of PEI Stocks]].
* DMEM/10% FBS/PSG and DMEM/10% FBS with no PSG
==Transfection Procedure==
* The morning of the transfection day, replace the media with fresh DMEM '''without PSG''' and containing 10 uL of 25 mM chloroquine (optional). Wait ~5h before going onto the next step.
* Prepare DNA in a sterile 1.5 mL tube or tubes as needed. Use 100 uL per well.
* Add 3 uL PEI per ug of DNA in OptiMEM. The total volume should be 100 uL per well. if transfecting multiple contstructs, make enough of this solution for all transfections.
* Gently add the PEI solution dropwise into the DNA solution (adding 100 uL to each 100 uL/well volume).
* Incubate at room temperature for 15-20min.
* Add transfection mixture slowly to the cells.
* Incubate overnight. The next day carefully replace with media containing PSG and/or treat as needed.
[[ Category: Cell Culture ]]
[[ Category: Tissue Culture ]]
[[ Category: Transfection ]]
[[ Category: Molecular Biology ]]
__NOTOC__
== Materials==
* Cells to transfect, generally growing in log phase at ~90% confluence
* Plasmids to transfect. Generally use 500 ng/well of a 6 well plate
* OptiMEM
* Chloroquine stocks (25 mM stocks). See [[Preparation of Chloroquine Stocks]].
* PEI (1 mg/mL stocks). See [[Preparation of PEI Stocks]].
* DMEM/10% FBS/PSG and DMEM/10% FBS with no PSG
==Transfection Procedure==
* The morning of the transfection day, replace the media with fresh DMEM '''without PSG''' and containing 10 uL of 25 mM chloroquine (optional). Wait ~5h before going onto the next step.
* Prepare DNA in a sterile 1.5 mL tube or tubes as needed. Use 100 uL per well.
* Add 3 uL PEI per ug of DNA in OptiMEM. The total volume should be 100 uL per well. if transfecting multiple contstructs, make enough of this solution for all transfections.
* Gently add the PEI solution dropwise into the DNA solution (adding 100 uL to each 100 uL/well volume).
* Incubate at room temperature for 15-20min.
* Add transfection mixture slowly to the cells.
* Incubate overnight. The next day carefully replace with media containing PSG and/or treat as needed.
[[ Category: Cell Culture ]]
[[ Category: Tissue Culture ]]
[[ Category: Transfection ]]
[[ Category: Molecular Biology ]]
__NOTOC__
