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.