SNAP circuit¶
This protocol describes the induction of the SNAP circuit in transient transfection in a 96-well plate.
Materials¶
Cells
Plates
DMEM supplemented with 10% FBS
PEI
Small molecule inducers:
Guanine (25 mM stock)
O6-benzylguanine (50 mM stock)
Solvent controls (e.g., 0.2N NaOH for guanine or DMSO for O6-benzylguanine)
Input plasmid: plasmid expressing SNAPtag conjugated with target protein (e.g., pHAGE-SNAP-tagBFP)
Output plasmid: plasmid expressing reporter gene-ribozyme switch cassette (e.g., pHAGE-UBC-mGreenLantern-p2g6)
Protocol¶
Day 1: Seed cells¶
Seed the cells at a density of 2.5-4e4 cells/well in a gelatin-coated 96-well plate.
Incubate the cells at 37ºC overnight.
Day 2: Transfection¶
Transfect 100 μg each of the input and output plasmids per well with PEI by following the general transfection protocol. To avoid cell death due to toxicity of the small-molecule inducers, reduce cell stress from transfection by using a lower PEI:DNA ratio of 3:1.
Incubate the cells at 37ºC overnight.
Day 3: Small molecule treatment¶
Dilute the small molecule inducers to the appropriate concentrations in the appropriate culture medium.
For both O6-benzylguanine and guanine, no greater than 100 µM should be used to minimize cytotoxicity.
Include conditions containing only the inducer solvent as a control (e.g., the same percent volume of DMSO only as in the O6-benzylguanine condition).
Remove the media from the wells, being careful not to detach the cells.
Add 100 μL/well of the inducer-containing media to the cells.
Incubate the cells for 48-72 hours at 37ºC.
Note
Be careful when changing the media for HEK293T cells—they easily detach, especially in a 96-well plate.
Day 5-6: Analysis¶
Analyze the cells via microscopy or flow cytometry.