Title |
Retrovirus Transfection Protocol |
Date Submitted |
April 24, 2012 |
Submitted by |
Berggren, Travis, tberggren@salk.edu |
Adapted from |
Salk STEM Cell Core in-house protocols |
Contributors |
Lutz, Margaret. Modesto, Veronica. Panopoulos, Athanasia. |
Affiliation |
The Salk Institute |
This protocol was developed to consistently produce a high titer retrovirus. This four-factor retrovirus can be used to reprogram fibroblasts into induced pluripotent stem cells.
Prepare 293T Cells:
Transfection of 293T Cells:
Product | Amount Needed | Supplier | Catalog Number |
---|---|---|---|
OPTI-MEM | 1 bottle | Life Technologies | 31985-070 |
Lipofectamine 2000 | 1ml vial | Life Technologies | 11668-01 |
50 ml conical tube | 8 | Corning | 430828 |
T175 flask | 4 | Corning | 431080 |
293T cells | 1 vial | ATCC | CRL-11268 |
293T media | See below |
293T Medium
Ingredient | Amount (mls) | Supplier | Catalog Number |
---|---|---|---|
IMDM | 85 | Life Technologies | 124400-53 |
FBS | 15 | Gemini or Various Sources**** | 100-106 |
NEAA | 1 | Life Technologies | 11140-050 |
Glutamax | 1 | Life Technologies | 35050-061 |
*Be very gentle when feeding 293T cells. They will detach from tissue culture treated plastic very easily. We found that culturing 293T cells in a T175 flask allowed us to produce a large amount of virus in a single vessel and to feed 293T cells more gently.
** When using our spinfection protocol there is no need to concentrate the virus.
*** In general, we like to combine the four factors together and aliquot into 4 mls. Each aliquot is then ready to reprogram one well of a 6 well plate when using our spinfection protocol.
****There is lot to lot variation with FBS that contributes to the overall efficiency of virus production using this protocol. It is advisable to screen multiple lots for efficiency of virus production. If lot screening is not an option, then HyClone sourced FBS is typically a safe bet.
This page was last modified on October 18, 2012