SNAP circuit (Transient expression in 96-well plate version)

Materials

  • Cells

  • Plates

  • DMEM supplemented with 10% FBS

  • PEI

  • Small molecule inducer such as guanine or O6-benzylguanine (check the concentration that you will need)

  • Output construct: plasmid expressing reporter gene-ribozyme switch cassette (e.g, pHAGE-UBC-mGreenLantern-p2g6)

  • Input construct: plasmid expressing SNAPtag conjugated with target protein (e.g, pHAGE-SNAP-tagBFP)

Procedure

Day 1: Seed cells

  1. Seed the cells at a density of 2.5-4k cells/well in a 96-well plate

  2. Incubate the cells at 37C overnight

Day 2. Transfection

  1. Transfect 100μg of the input and output plasmids per well with PEI by following the general transfection protocol. If your small molecule or the solvent has cytotoxicity, using a lower PEI:DNA ratio of 3:1 is recommended.

  2. Incubate the cells overnight

Day 3: Small molecule treatment

  1. Remove the media from the wells, being careful not to detach the cells, and add 100μl of fresh media that contains the small molecule inducer such as O6-benzylguanine or the solvent.

  2. Incubate the cells for 48-72 hours at 37℃

Note

Be careful when changing the media for 293T cells—they easily detach, especially in a 96-well plate.

Day 5-6

Analyze the cells via microscopy or flow cytometry.